University of Southern California Dissertations and Theses

The biochemistry of glycoproteins from human prostate

(USC Thesis Other)

The biochemistry of glycoproteins from human prostate

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content THE BIOCHEMISTRY OF GLYCOPROTEINS
FROM HUMAN PROSTATE
by
Thaddeus G eorge Pullano
A D issertation P resented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a rtial F ulfillm ent of the
R equirem ents for th e D egree
DOCTOR OF PHILOSOPHY
(Biochem istry)
O ctober 1983
UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SC H O O L
UNIVERSITY PARK
LOS A N G ELES, C A L IF O R N IA 9 0 0 0 7
This dissertation, written by
. -Tha-ddeus. .Gsp.Ege. .Eullauo.
under the direction of his..... Dissertation Com -
mittee, and approved by all its members, has
been presented to and accepted by The Graduate
School, in partial fulfillment of requirements of
the degree of
D O C T O R O F P H I L O S O P H Y
f*r
Dean
DISSERTATION COMMITTEE
$ - 7ft
DEDICATION
This d issertatio n is dedicated to my
wife, Sheila M arie Pullano, whose
unending love, support, encourage
m ent and p atien ce m ade this en
deavor possible.
ii
ACKNOWLEDGEMENTS
I am gratefu lly indebted to Dr. Zoltan A. Tokes for offering his guidance
and support and for providing the laboratory space and funding for my
research. Special thanks to Dr. Luisa R aijm an and Dr. R ichard Wood for
finding tim e in th eir busy schedules to be on my thesis com m ittee. My
appreciation is extended to Dr. G erry D erm er and Dr. Larry Silverm an for
tissue and fluid sam ples and th eir collaboration. Thanks also to Dr. Clive
Taylor for collaboration and for allowing me to squeeze into his busy
laboratory. I also app reciate Dr. Wesley N aritoku's discussions and interesting
tissue sections. I am g ratefu l to Dr. A shraf Imam for introductions to
im m unohistochem istry and the fundam entals of hybridom a technology, as well
as many hours of fru itfu l discussions. Thanks also to Dr. Anthony Neri for
inform ation and advice on cell cu ltu res and photom icrography, and for many
in teresting conversations. Thanks to Dr. William Shell for sam ples and
collaboration. I am indebted to Mr. Csaba Csipke for his tech n ical expertise in
cell culture. Thanks go to Ms. Ruth Olivo for technical assistance and to Ms.
Ginny K ortes, R.N ., for th e m any tissue and fluid sam ples she provided.
Finally, I am deeply indebted to my w ife, Sheila, and h er p aren ts, Mr. and Mrs.
Ralph M athews, for th eir encouragem ent, support and pray ers during graduate
school.
iii
TABLE OF CONTENTS
Page
DEDICATION................................................................................................... ii
ACKNOWLEDGEMENTS............................................................................. iii
LIST OF FIGURES........................................................................................... v
LIST OF TABLES........................................................................................... viii
GLOSSARY...................................................................................................... ix
INTRODUCTION ........................................................................................... 1
CHAPTER I : M aterials and M ethods............................................ 10
CHAPTER H : Analysis of P roteins and G lycoproteins in P ro
sta tic Fluid Using Two D im ensional Gel Elec
trophoresis, W estern Blots, and Im m unoauto-
ra d io g ra p h y .................................................................. 38
CHAPTER HI: C reatin e Kinase-BB in P ro static Fluid. P ar
tia l P u rification and Identification on Two
D im ensional Gels of P ro sta tic F lu id .................... 67
CHAPTER IV : Analysis of the G lycoproteins Synthesized by
Benign H yperplastic P ro sta te Organ C ultures
and by P ro sta te C arcinom a Cell Line PC-3 . . 84
CHAPTER V : Serum a -l-A c id G lycoprotein. Analysis of
Isotype H eterogeneity in Norm al and D isease
S tates by Two Dim ensional Gel E lectropho
resis ................................................................................ 109
CHAPTER V I: C onclusion...................................................................... 1 3 9
REFERENCES................................................................................................... 146
iv
LIST OF FIGURES
Page
2-1 Double im m unodiffusion of p ro sta tic fluid and serum
against antibodies to serum p ro te in s ..................................... 47
2-2 2D gel electrophoresis of 30 p.1 of p ro sta tic fluid.............. 47
2-3 2D gel resolution of the p ro tein s in 5 (J .1 of p ro static
fluid using IEF pH range 5 to 7.................................................. 49
2-4 2D gel resolution of th e p ro tein s in 20 p.1 of p ro static
fluid using IEF pH range 5 to 7.................................................. 49
2-5 2D gel electrophoresis of 5 p.1 of p ro sta tic fluid, stained
by periodic acid Schiff’ s re a c tio n .............................................. 51
2-6 2D gel electrophoresis of 10 J J .1 of norm al serum pro
teins, using IEF pH range 5 to 7 ................................................ 51
2-7 The location of a-1 -p ro tein ase inhibitor on 2D e le c tro
phoretic gels as determ ined by im m unoautoradiography
o f a W estern b l o t ............................................................................. 53
2-8 Com posite map localizing a-1 -p ro tein ase inhibitor by
superim posing W estern blo t autoradiograph over
Coom assie B lue-stained p ro tein p a tte rn ................................. 53
2-9 2D gel electrophoresis of p ro sta tic fluid using pH range 4
to 6 for I E F ........................................................................................ 55
2-10 C om posite map localizing haptoglobin $-chain on 2D gels
by superim posing a p ro sta tic fluid W estern blot auto
radiograph over a p ro sta tic fluid p ro tein p a tte rn ................. 55
2-11 L ocalization of a -1 -acid glycoprotein on a W estern blot
of norm al serum by im m unoautoradiography......................... 57
2-12 2D gel electrophoresis of p ro sta tic fluid using pH range
of 3.5 to 5 for I E F ........................... 57
3-1 Sephadex G-100 colum n chrom atography of p ro sta tic
f lu id .................................................................................................... 74
3-2 D ensitom etry of CK isozym es a fte r electrophoresis on
agarose f ilm ....................................................................................... 74
v
P age
3-3 DEAE-cellulose chrom atography of activ e CK fractions. 76
3-4 2D gel resolution of radioiodinated CK-BB p reparation
containing A G P ............................................................................... 76
3-5 2D gel resolution of purified CK-BB p r o te in ........................ 78
3-6 2D gel electrophoresis of radioiodinated C K -B B .................. 78
3-7 2D gel electrophoresis of 20 |J.l of p ro sta tic fluid, using
pH range 5 to 7 for IEF.................................................................. 80
14
4-1 R esolution of ( C )-glucosam ine labeled glycoproteins,
released from BPH organ culture, by 2D gel electropho
resis ...................................................................................................... 97
14
4-2 Com posite map of ( C )-glucosam ine labeled glycopro
teins, released from BPH organ cultures, resolved by 2D
gel e le c tro p h o re sis........................................................................ 97
14
4-3 R esolution of ( C )-glucosam ine labeled glycoproteins,
released from p ro sta te carcinom a cell line PC-3, by 2D
gel e le c tro p h o re sis........................................................................ 99
14
4-4 Com posite map of ( C )-glucosam ine labeled glycopro
teins released from PC-3 cells, resolved by 2D gel
e le c tro p h o re sis................................................................................ 99
4-5 2D gel resolution of serum proteins, using pH range 4 to
6 for I E F ............................................................................................ 101
4-6 2D gel electrophoresis resolving acidic glycoproteins
released by PC-3 c e lls .................................................................. 101
4-7 Im proved 2D gel resolution of acidic glycoproteins re
leased by PC-3 cells in c u ltu r e .................................................. 103
4-8 Com posite map com paring 2D gel resolution of acidic
glycoproteins released by PC-3 cells and 2D gel resolu
tion of serum a -1 -a cid g ly c o p ro te in ....................................... 103
5-1 2D gel resolution of a-1 -a cid glycoprotein from serum of
a healthy, norm al v o lu n te e r....................................................... 122
5-2 2D gel resolution of AGP tre a te d w ith neuram inidase . . . 122
5-3 A utoradiographs of 2D try p tic peptide m a p s ........................ 124
5-4 T hree layer peroxidase antiperoxidase staining of fo r
m alin fixed BPH tissue section using anti-A G P antibodies 126
vi
Page
5-5 Three layer peroxidase antiperoxidase staining of fo r
m alin fixed lymph node section containing m e ta sta tic
p ro sta te carcinom a, using anti-A G P a n tib o d ie s................. 126
5-6 2D gels of serum , pH range 3.5 to 5, from norm al
individual and p ro sta te carcinom a p a tie n t............................ 128
5-7 Bar graph illu stratin g re la tiv e distribution of AGP pro
tein among isotypes in norm al individuals, BPH and CAP
p a tie n ts .............................................................................................. 130
LIST OF TABLES
P age
3-1 L ectin affin ity chrom atography of C K -B B ............................. 73
4-1 BPH organ culture and PC-3 cell culture biosynthetic
products th a t share iso electric points and m olecular
w e ig h ts................................................................................................. 93
4-2 BPH and p ro sta tic fluid com ponents th a t share isoelec
tric points and m olecular w e ig h ts............................................ 94
14
4-3 Im m unoprecipitations of ( C )-glucosam ine labeled gly
coproteins from su pernatants of BPH organ cu ltu res . . . 95
14
4-4 Im m unoprecipitations of ( C )-glucosam ine labeled gly
coproteins released into cu lture medium by PC-3 cells,
and control im m u n o p recip itatio n s............................................ 96
5-1 C h aracteristics of norm al serum a -l-a c id glycoprotein
iso types resolved on 2D g e ls ....................................................... 119
5-2 R eproducibility in determ ining the p ercen tag es of AGP
iso ty p e s ............................................................................................... 120
5-3 P ercen t distribution of AGP is o ty p e s..................................... 121
viii
GLOSSARY
Achy
ADP
AGP
ATP
BB
BIS
BPH
BSA
°C
CAB
CAP
CK
CK-BB
CK-B
cm
Con A
cpm
2D gels
DEAE
dpm
EC 2.7.3.2
g
G-6-PDH
GARGG
a -1-an t ichy m o tr yp sin
adenosine diphosphate
a -l-a c id glycoprotein
adenosine triphosphate
creatin e kinase isozym e, brain type
N,N’ -m ethylene-bis-acrylam ide
benign p ro sta te hyperplasia
bovine serum album in
degrees centigrade
carcinom a of the b reast
carcinom a of the p ro sta te
creatin e kinase
the dim er, c re atin e kinase isozym e BB
single subunit of CK-BB
c en tim eter
C oncanavalin A
counts p e r m inute
two dim ensional iso electric focusing
SDS-polyacrylam ide gel electrophoresis
diethyl am inoethyl
d isintegerations p e r m inute
Enzyme Com m ission no. 2.7.3.2, creatin e kinase
gravity, 9.8 m eters p e r second
glucose-6-phosphate dehydrogenase
goat an ti-rab b it gam m a globulin
IX
gp 42 PC-3 acidic glycoprotein of 42,000 m olecular w eight
Hp haptoglobin
h r hour
IEF iso electric fochsing, the 1st dim ension of 2D gels
IgA im m unoglobulin type A
IgG im m unoglobulin type G
IgM im m unoglobulin type M
kd kilodaltons
LAC-USC Los A ngeles C ounty-U niversity of Southern C alifornia
\ i d m icro Curie
m icrogram
|il m icro lite r
M m olar
MB
cre atin e kinase isozym e, h e a rt type
m Ci m illi C urie
mg m illigram
ml m illiliter
mm m illfm eter
raM milli m olar
MM
c re atin e kinase isozym e, m uscle type
mU
m illiunit
min m inute
N norm al
NAD
nicotinam ide adenine dinucleotide
NADH
nicotinam ide adenine dinucleotide, reduced form
NADP
nicotinam ide adenine dinucleotide phosphate
NANA N -acety l neuram inic acid, a sialic acid
nm nanom eters
NP-40 nonionic d e terg en t Nonidet P-40
OD optical density
PA P horseradish peroxidase-antihorseradish peroxidase
com plex
PAS peroidic acid-Schiff's
PBS phosphate bu ffered saline
PC-3 th e PC-3 p ro sta tic carcinom a cell line
PF49 p ro sta tic fluid com ponent w ith 49,000 m olecular w eight
PF52 p ro sta tic fluid com ponent w ith 52,000 m olecular w eight
Pi
iso electric point
PI
a-1 -p ro tein ase inhibitor
p ro tein A cell wall p ro tein isolated from Staphylococcus aureus
Cowan I strain
PTA phosphotungstic acid
SDS sodium dodecyl su lfate
SDS-PAGE
SDS-polyacrylam ide gel electrophoresis, the 2nd
dim ension o f 2D gels
Staph A Staphylococcus aureus
TBS Tris-H Cl b u ffered saline
TBS/BSA Tris-H Cl b uffered saline containing bovine serum album in
TCA trich lo ro acetic acid
TEMED
N jN jN ^N '-tetram ethylethylenediam ine
Tris
Tris(Hydroxym ethyl) am inom ethane
Tris-f3ME Tris-H C l b u ffer containing $ -m ercap to eth an o l
U unit
v/v
volum e to volum e ratio
xi
w /v
WGA
w eight to volum e ra tio
w heat germ agglutinin
xii
INTRODUCTION
The p ro sta te is a ch estn u t sized organ lo cated a t the base of th e penis,
im m ediately below th e bladder. It consists of a fibrom uscular stro m a
surrounding tubuloalveolar glands, all enclosed w ithin a connective tissue
capsule. The p ro sta te 's function is to se c re te a thin, milky, slightly alkaline
fluid which m akes up a portion of the sem en. This secreto ry a ctiv ity depends
on te sto ste ro n e which is supplied via th e blood from the te stes. P ro sta tic
secretio n s e n ter the u re th ra , which the gland surrounds, by way of a series of
sec re to ry ducts.
A denocarcinom a and benign hyperplasia of the p ro sta te usually occur in
older m ales, and increase in incidence w ith advancing age. Autopsy studies
have shown th a t p ro sta te carcinom a is rarely found before the age of 50.
However, by age 70 about 50% of A m erican m ales have some form of this
disease (Scott e t al., 1969; Franks, 1977). P ro sta te carcinom a is o ften la te n t,
showing no clinical sym ptom s throughout an individual's lifetim e. Some la te n t
carcinom as, for unknown reasons, becom e clinically m anifest. O ften, p ro sta te
carcinom a is not diagnosed u n til m e ta sta ses have occurred and the disease has
advanced to an incurable stag e (Parry, 1975; G rayhack e t al., 1980). As a
result, this carcinom a is the second m ost com m on cause of m ale cancer d eaths
in th e U nited S ta te s (Silverberg, 1982). Physical exam ination of th e p ro sta te
is d ifficu lt due to its relativ ely inaccessible location a t the base of the penis.
In the p a st, the m ajor clinical te st for neoplasia has been d ig ital exam ination
of the gland through the re c ta l wall. This has lim ited value in screening early
m alignancies since a sm all lump deep w ithin th e gland can be overlooked by a
physician's probing finger. Hudson e t al., (1954) found th a t only 9 o f 39
1
p a tie n ts w ith histologically confirm ed carcinom a had a stoney, hardened area of
the p ro sta te th a t was readily d etectab le by d ig ital exam . More recently,
colorom etric te sts or radioim m unoassays d etectin g elev ated levels of the
p ro sta te specific enzym e p ro sta tic acid phosphatase in p a tie n t serum have
gained use as a screening procedure for p ro sta te cancer candidates. E levated
serum p ro sta tic acid phosphatase or a hardened nodule on the p ro sta te are
highly suspicious, b u t inconclusive. Diagnosis m ust still be confirm ed by
biopsy and histologic evaluation.
The ex ten t of carcinom a involvem ent is crucial to the p a tie n t’s prog
nosis. If the disease has widely m etastasized, prognosis is poor and therapy is
m erely p alliative. If th e carcinom a can be d e tec te d a t an early stage, while
entirely w ithin the p ro sta tic capsule, the p a tie n t can be tre a te d effectiv ely by
rad ical surgery w ith good p o te n tia l for cure. B iochem ical investigation of the
p ro sta te may lead to im proved m ethods o f early detectio n .
N orm al glandular tissue, such as p ro sta te, has a ch arac te ristic , well
organized stru ctu re in which ep ithelial cells line the acini and surround the
acinar lumina. A pocrine and m erocrine secretions of th e ep ithelial cells are
released into the lum ina w here they are sequestered from o th er body com part
m ents, such as th e blood circu lato ry system . From the lumina, the secreto ry
products are channeled into ducts and are u ltim ately ex creted into the u re th ra
to becom e p a rt of th e sem inal fluid. Tissue from epith elial benign p ro sta te
hyperplasia has an a rc h ite c tu re much like norm al p ro sta te . There is epithelial
cell overgrow th into lum inal spaces, but the acini are in ta c t and invasive
grow th into surrounding strom a does not occur. Therefore, hyperplastic tissue
may be expected to m aintain th e separation of secreto ry products from other
body com partm ents. However, in adenocarcinom as, p articu larly poorly d iffer
2
e n tia ted types, the norm al th re e dim ensional stru ctu re is not m aintained. Cells
are poorly organized and acini are e ith e r rudim entary or en tirely absent.
N orm ally sequestered products may co llect in cells or residual acini and leak
into surrounding connective tissue, eventually finding th eir way into blood
circulation (Brandes and K ircheim , 1977). A search for such m olecules in the
blood stream has p o te n tia l for diagnostic u tility (Tokes e t al., 1983). A first
approach tow ards exam ining this hypothesis is an analysis of com ponents th a t
m ake up the norm al glandular secretion. G lycoproteins are well suited for
study due to th eir physical c h a ra c te ristic s which make biochem ical m anipula
tions, such as electrophoresis, easy. A dditionally, th eir stab ility in many
biological m ilieu, including blood, increases th eir p o te n tia l as useful organ
m arkers. Finally, glycoproteins are easily radiolabeled by in vitro culturing of
cells or organ fragm ents in th e p resen ce of radiolabeled m etabolic precursors.
T herefore, experim ents can be perform ed to com pare the biosynthetic products
of p ro sta te cu ltures w ith the com ponents in p ro sta tic secretion.
A gland's ep ithelial cells produce unique com ponents. For exam ple, b re a st
ep ith elial cells are known to specifically synthesize casein and a-lactalb u m in
(Laurence, 1978). It is th erefo re assum ed th a t the p ro sta te sim ilarly produces
unique com ponents. The p ro sta tic secretion, or p ro sta tic fluid, co n stitu tes 30%
to 40% of the ejacu late volum e (for review s see Eliasson, 1976; K halil e t al.,
1979). Previous rep o rts have analyzed p ro static fluid expressed by d igital
m assage o f the gland. No d ifferen ces w ere found in to ta l p ro tein levels when
com paring norm al and pathologic specim ens (Soanes e t al., 1963; Resnick and
Stubbs, 1978). Electrophoresis on various support m atrices w as perform ed in
a tte m p ts to define p ro sta tic fluid pro tein p a tte rn s th a t may co rrelate w ith
disease. N ylander (1955) first sep arated p ro sta tic fluid into th ree fractions
3
having electro p h o retic m igrations equivalent to alpha, b eta and gam m a glob
ulins of serum . He d e tec te d an increase in the alpha fractio n o f p ro sta tic fluids
from carcinom a and benign hyperplasia p atie n ts as com pared to norm al.
P ro sta titis sam ples showed increases in the gam m a fractio n . The com plexity of
p ro sta tic fluid p ro tein s becam e m ore evident by electrophoresis on agar.
Soanes e t al., (1963), resolved nine com ponents and found th a t p a tte rn s in
pathological fluids d iffered from norm al fluids. However, the abnorm al
p a tte rn s w ere poorly resolved and did not c o rre la te w ith disease s ta te . Denis
e t al., (1962), d e tec te d th irteen p ro teins by agar gel electrophoresis o f pro static
fluids from benign hyperplasia and carcinom a p atie n ts and identified several
bands in each of the prealbum in, alpha, b e ta and gam m a regions. These
investigators saw an increase in the slow est band, corresponding to gam m a
globulin, in fluids from adenocarcinom a p atien ts. Both Soanes et al., and
Denis e t al., found a com ponent w ith m igration equivalent to album in.
One dim ensional polyacrylam ide gel electrophoresis of p ro sta tic fluid has
b een p erform ed under b o th reducing and non-reducing conditions. In a non
reducing b u ffer, fifteen p ro tein bands w ere identified (A tanasov and Gikov,
1972). In the presence o f sodium dodecyl su lfate (SDS) under reducing
conditions, p ro static fluid p ro tein s w ere resolved into six m ajor and several
m inor bands by Resnick and Stubbs (1978). No differen ces w ere d e tec te d
betw een benign hyperplasia and carcinom a specim ens as determ ined by densito-
m etric scanning of stained gels. B alerna e t al., (1982), resolved ten m ajor
bands when p ro sta tic fluid was analyzed by one dim ensional SD S-electropho-
resis, five of which stained as glycoproteins. They discovered distin ct d iffe r
ences betw een p ro sta tic fluids of norm al and p ro sta titis p atien ts. Iso electric
focusing also was perform ed, b u t the resulting bands w ere poorly resolved as
4
com pared to those obtained by SD S-electrophoresis. Two dim ensional gel
electrophoresis of p ro sta tic fluid has been perform ed (D erm er e t al., 1982;
C a rte r and Resnick, 1982) and some of the rep o rted com ponents appear to be
the sam e as those described in C hapter II. However, no a tte m p t has been made
to identify im m unologically the bands or groups of p ro tein s and glycoproteins
resolved by any o f the above m ethods.
O ther biochem ical observations have been m ade on expressed p ro sta tic
fluids. Polyam ines, zinc, and cholesterol levels w ere determ ined in fluids from
norm al, benign hyperplasia, carcinom a and p ro sta titis p a tie n ts b u t no co rrela
tion w ith p ro sta tic pathology was found (Anderson and F air, 1976). An
im m unochem ical search for specific serum p ro tein s in p ro sta tic fluid showed
insignificant elevations in im m unoglobulins A, G and M in p a tie n ts w ith p ro sta te
carcinom a as com pared to hyperplasia and p ro sta titis. However, tran sferrin
and com plem ent com ponents C3 and C4 w ere significantly elev ated in c a r
cinom a as com pared to the oth er diseases when determ ined by radial im m uno
diffusion (Grayhack e t al., 1979)* No a tte m p ts w ere made to establish w hether
these p ro tein s w ere produced by the p ro sta te gland or derived from blood.
Assays of enzym es in p ro sta tic fluid have shown som e co rrelatio n w ith
p ro sta te disease. L a c tate dehydrogenase (LDH) was found in both p ro sta tic
tissue and fluid (Denis e t al., 1962). When p ro sta tic fluid was sep arated
electrophoretically, LDH resolved into five d istin ct isozym es. The ra tio of
isozym e 1 to isozym e 5 was found to increase in p ro sta tic carcinom a b u t not in
benign hyperplasia (Grayhack e t al., 1977b). This co rrelatio n has p o te n tia l as a
useful disease indicator.
Acid phosphatase was first found to be elevated in serum of m e ta sta tic
p ro sta te cancer p a tie n ts by G utm an and cow orkers (Gutm an, Sproul and
5
G utm an, 1936; G utm an and Gutm an, 1938). In v estig ated as a p o te n tia l p ro sta te
m arker, this enzym e is p resen t in high levels in the p ro sta te . P ro static fluid
h as been te ste d for acid phosphatase activ ity (Kent e t al., 1970; G rayhack e t
al., 1977a). It's levels in the fluid w ere found to be decreased in carcinom a,
increased in benign hyperplasia and g reatly increased in p ro sta titis. P ro sta tic
acid phosphatase has been extensively studied and appears to be antigenically
d ifferen t from serum acid phosphatase and from a ubiquitous lysozom al acid
phosphatase. In addition, it has been im m unologically localized in p ro sta te
ep ith elial cells (Choe e t al., 1980). It has a subunit m olecular w eight of about
45,000 to 50,000 dal tons (Choe e t al., 1978). It also expresses heterogeneous
carbohydrate content as re fle c te d by m ultiple isozym e form s under isoelectric
focusing conditions (Ostrow ski e t al., 1970). As an im m unological m arker in
serum and tissue, p ro static acid phosphatase holds g re a t p o te n tia l in future
clinical testin g for p ro sta te carcinom a (Herschm an, 1980; Choe and Rose,
1982).
P ro static fluid was found to contain high con cen tratio n s of c reatin e
kinase isozym e BB (Silverm an e t al., 1979). This enzym e was also seen in
epith elial elem ents of the p ro sta te by im m unohistochem istry (Silverman et ah,
1979; Pretlow e t al., 1982). The am ount of CK-BB in p ro sta te carcinom a tissue
decreased when com pared to hyperplastic tissue (Pretlow e t aL, 1982) and
increased in the sera of som e p ro sta te carcinom a p atien ts, especially those w ith
m etastases (Feld and W itte, 1977; Silverm an e t al., 1979? Form an, 1979; Feld
e t al., 1980). The d ata suggest a co rrelation of elev ated serum CK-BB w ith the
ex ten t o f disease which may have p red ictiv e value in p ro sta te carcinom a
p atien ts.
6
P ro sta te antigen is a recen tly discovered p ro state-asso ciated com ponent
described by Chu and cow orkers (Wang e t al., 1979). It is found in p ro sta tic
fluid and is im m unologically d e tec te d in p ro sta te d uctal epithelial cell cyto
plasm (Papsidero e t al., 1981; Wang e t al., 1982). It appears p ro sta te specific
and is d e tec ta b le in the serum . P ro sta te antigen is a glycoprotein w ith
m olecular w eight of 33,000 to 34,000 daltons which displays m ultiple iso electric
isom ers o f p i 6.8 to 7.5 (for review see Wang e t al., 1982). Prelim inary re p o rts
show th a t p ro sta te antigen m ay be useful as a p ro sta te m arker since it is
elevated in serum of p ro sta te carcinom a p a tie n ts, especially in advanced stages
of the disease (K uriyam a e t al., 1980). It may y e t b e classified as an enzym e
m arker, since p roteinase a ctiv ity has been found associated w ith the purified
m olecule (Ban et al., 1983).
To d ate, screening te sts for p ro sta te carcinom a, even th e prom ising te sts
for p ro sta tic acid phosphatase and p ro sta te antigen, are b est able to identify
late stag es of the disease. There is still a need for new m arkers and m ore
sensitive assays to discover the e arliest carcinom a stages. The studies rep o rted
here describe an approach tow ards ch aracterizin g new p ro sta te m arkers th a t
may u ltim ately prove useful in developing an assay or a b a tte ry of assays to
d e te c t carcinom a of th e p ro sta te .
The objectives of this study w ere:
1. To c h arac te riz e th e m ajor norm al p ro sta te glandular products, found in
hum an p ro sta tic fluid, as the first step in a search for p o te n tia l organ
m arkers. A useful m ethod for analyzing the com plex m ixture of p ro tein s
found in p ro sta tic fluid is two dim ensional SDS-polyacrylam ide gel elec
trophoresis (2D gels).
7
2. To identify which of th e m ajor p ro tein s found in p ro sta tic fluid, if any,
are related to serum proteins.
3. To determ ine if bio synthesized glycoproteins produced by hum an p ro sta te
organ cu ltu res have sim ilarities w ith norm al, m ajor p ro sta tic fluid com po
nents.
4. To in v estigate w hether the biosynthesis of any of th e organ cu lture
products is m aintained in v itro by a hum an p ro sta te carcinom a cell line
derived from a m e ta sta tic tum or.
5. To determ ine w hether the localized changes of p ro sta tic carcinom a resu lt
in a system ic change d e te c ta b le in th e blood.
The first objectives are aim ed tow ards describing p o te n tia l p ro sta te
m arkers and w ere form ulated according to the following hypothesis. If the
synthesis of p ro sta te products is m aintained by p ro sta te organ in vitro, as well
as by p ro sta te adenocarcinom a cells in vitro, th ere is an increased likelihood
th a t these products will be lost from th e tum or site in vivo, due to disrupted
tissue stru ctu re, and becom e d e tectab le in serum . C hapter II deals w ith the
delineation of glycoproteins in p ro sta tic fluid and id en tificatio n of blood serum
p ro tein s p resen t in the fluid. C hapter m describes a schem e for the p a rtia l
pu rificatio n of c re a tin e kinase BB, and its localization on 2D gels. C hapter IV
d etails the investigations of glycoprotein biosynthesis by p ro sta te organ cul
tu res and the p ro sta te carcinom a cell line PC-3.
The last objective was to search for gross system ic changes, d etectab le in
th e blood, th a t c o rre la te w ith p ro sta tic carcinom a. None of the p ro sta tic
glycoproteins described in the following chapters are found in serum when
te sted by the relativ ely insensitive 2D gel assays used in these studies.
8
However, an in terestin g observation was m ade on a liver product, the acute
phase re a c ta n t a -l-a c id glycoprotein. This serum com ponent resolves into
several isotypes on 2D gels, and the isotype p a tte rn d iffers in p a tie n ts w ith
p ro sta te disease as com pared to norm al individuals. This work is discussed in
C hapter V.
It is my hope th a t the work rep o rted here will aid in understanding the
local and system ic changes th a t occur in p a tie n ts w ith p ro sta te cancer, and will
eventually lead to early disease d etectio n and increased cure ra te s in p a tie n ts
suffering from this illness.
9
CHAPTER I
MATERIALS AND METHODS
10
M aterials
C hem icals. BioRad L aboratories, Richm ond, CA, was the source of
acrylam ide (99.9%), BIS (N ,N '-m ethylene-bisacrylam ide), riboflavm -5-phos-
p hate, TEMED (N,N, N 'jN '-tetram ethylethylenediam ine), sodium dodecyl sul
fa te (SDS, 99%), urea, low and high m olecular w eight standards for SDS-
electrophoresis, and p ro tein assay kit w ith bovine gam m a globulin standard.
N onidet P-40 (NP-40) was purchased from D ata P a rticle Labs, Lim ited,
Elm hurst, IL. All photographic chem icals and ft-m ercaptoethanol w ere from
E astm an Kodak Company, R ochester, NY. Sigma C hem ical Com pany, St.
Louis, MO, was supplier of bovine serum album in (BSA), SDS (95%, suitable for
slab gel electrophoresis), Tris-base, glycine, agarose (medium electroendos-
mosis), ammonium p ersu lfate, brom ophenol blue, Coom assie B rilliant Blue R,
Schiff's reagent, neuram inidase type Vm from Clostridium perfringens, and
high m olecular w eight standards for SD S-electrophoresis. A m phohnes w ere
purchased from LKB, P leasant Hill, CA. Flow L aboratories, Rockville, MD,
was the source of RPMI 1640 medium, fe ta l bovine serum (FBS), insulin,
fungizone, penicillin, streptom ycin and gentam ycin. Glucose, Medium 199, and
minimum essen tial m edium (MEM) w ere from Grand Island Biological C orpor
ation, Grand Island, NY. Trypsin, 3X crystalized, was from W orthington
B iochem ical C orporation, Freehold, N J. C albiochem -Behring, San Diego, CA,
was the source of phenylm ethylsulfonyl fluoride and fixed Staphylococus
aureus cells of Cowan I strain . M olecular w eight standards for column
chrom atography w ere from Boehringer Mannheim B iochem icals, Indianapolis,
IN. P urified hum an a - l- a c id glycoprotein was a kind g ift of the DAKO
C orporation, Santa B arbara, CA. L ectin affin ity m atrices w ere g ifts from Dr.
11
------------------------------------------------------- --------------------------------------- 125
A. Im am . M allinckrodt, Inc., Paris, KY, kindly provided hum an ( I)-creatin e
kinase-BB. All other lab o rato ry chem icals w ere of th e highest p u rity
available. Solutions w ere m ade w ith double distilled w ater.
R adioisotopes and re la te d supplies. New England N uclear C orporation,
14
Boston, MA, was the source of D - [ C(U) ]-glucos am ine hydrochloride (250-
o c
350 m C i/m m ol, in 9:1 ethanol: H^O), L- [ S]-m ethionine, (1200 C i/m m ol),
Protosol p ro tein solubilizing solution, Econofluor scin tillatio n cocktail, and
125
EnHance fluorography enhancer. C arrie r fre e Na ( I) (15.9 m C i/ ng of
iodine), PCS-II and ACS scintillation cocktails for aqueous solutions w ere
purchased from A m ersham C orporation, A rlington H eights, IL. E astm an
Kodak Com pany, R ochester, NY, was the source of X -om at AR X -ray film , X-
ray exposure holders and film processing chem icals. C ronex intensifying
screens w ere from E.I. DuPont Nemours and Com pany, W ilm ington, DE.
S cintillation counters w ere th e D elta 300 from Searle A nalytic, Incorporated,
Des Plains, IL, Beckm an 9000, Beckm an C orporation, Fullerton, CA, and
PRIAS PLD T ri-C arb, P ackard Instrum ent Com pany, Downers G rove, IL.
G am m a C ounting was done on a P ackard A uto-G am m a Scintillation Spectro
m e te r.
Im m unochem icals. Dako C orporation, Santa B arbara, CA, was the
source of rab b it antibodies recognizing th e following hum an com ponents: a-1-
acid glycoprotein, a-l-an tich y m o try p sin , a-1 -p ro tein ase inhibitor (a -l-a n ti-
trypsin), haptoglobin, and whole serum proteins. As well, Dako was supplier of
swine a n ti-ra b b it im m unoglobulin and PAP (horseradish peroxidase - anti
horseradish peroxidase com plexes). N orm al swine serum was from Flow
12
L aboratories, H aw thorne, CA. D iam inobenzidine was obtained from E lectron
M icroscopy Sciences, F ort W ashington, PA. Perm ount was from Fischer
S cientific, F air lawn, N J. Sheep anti-hum an creatin e kinase BB w as from E.
M erck Biochem icals, D arm stadt, F.R .G . Calbiochem -B ehring was the source
of rabbit anti-hum an a - l- B glycoprotein, rabbit anti-hum an a-2-H S glycopro
tein, and goat an ti-rab b it gam m a globulin. Im m unodiffusion p la te s and well
c u tte rs w ere from Miles L aboratories, Elkhart, IN.
Form alin fixed Staphylococcus aureus cells, Cowan I strain, w ere ob
tained from eith er C albiochem -B ehring or B ethesda R esearch L aboratories,
R ockville, MD. P urified p ro tein A was from Sigma C hem ical C orporation.
M iscellaneous supplies and apparatus. N itrocellulose sheets, type HA,
0.45 m icron pore size, was purchased from M illipore C orporation, Bedford,
MA. P h arm acia Fine C hem icals, P iscataw ay, N J, was the source of cyanogen
brom ide activ ated Sepharose 4B, and Sephadex types G-25, G-100, and G-200.
Sigma C hem ical C orporation was supplier of diethylam inoethyl (DEAE)-cellu-
lose. Disposable p lasticw ares for tissue culture w ere from Corning Glass
Works, Corning, NY, or Falcon P lastics, Becton-D ickinson Lab w are, Oxnard,
CA. Cellulose thin layer chrom atography sheets w ere purchased from
E astm an Kodak Company. C hrom ist spray unit was from G elm an Instrum ent
Company, Ann Arbor, MI. Am icon C orporation, Lexington, MA was the source
of stirre d u ltra filtra tio n cells, Models 52, 12, and 3, as well as m em brane types
PM and YM. N egative pressure d ialysis-concentration apparatus was the
ProD iCon m odel 320 from Bio-M olecular Dynamics, B eaverton, OR. Brinkman
Instrum ents, W estbury, NY was the m an u factu rer of Polytron hom ogenizer.
Tube gel electrophoresis cell Model 150A w as purchased from BioRad
13
L aboratories, Richm ond, CA. H oefer S cientific Instrum ents, San Francisco,
CA, was th e supplier of the following: Model SE500 slab gel electrophoresis
u n it, SE540 slab gel dryer, and the TE42 transphore apparatus for W estern
blots. Pow er supply was Buchler 3-1500, Buchler Instrum ents, F o rt Lee, N J.
F lat bed electrophoresis apparatus was the M ultiphor unit from LKB, P leasant
Hill, CA. D en sito m eter was Model bm i TRD-85 from Brumac Industries,
H untington Beach, CA. R adiom eter conductivity m e te r was from The London
Com pany, C leveland, OH. M icro-com bination pH probe was purchased from
M icroelectronics, Inc., Londonderry, NH.
M ethods
P ro sta tic fluid. P ro sta tic fluids w ere obtained from fresh hum an pros
ta te s im m ediately a fte r to ta l p ro sta tec to m y . The glands w ere sliced w ith a
scalpel blade and fluid was co llected th a t accum ulated a t cu t surfaces.
Samples w ere cen trifu g ed a t 5°C for 10-15 m inutes, 500 x g, to rem ove debris.
S upernatants w ere stored a t -20°C .
Serum co llectio n . Blood serum specim ens w ere obtained by the Oncology
R esearch Nursing Team a t the U niversity of Southern C alifornia C om prehen
sive C ancer C enter. Diagnosis of benign or can cer disease was confirm ed by
pathological rep o rts. P reo p erativ e sera w ere co llected 24 hours p rio r to
surgery, and p o sto p erativ e sera w ere co llected 24 to 96 hours a fte r surgery.
O ther p a tie n t sera w ere obtained during routine exam ination. N orm al sera
w ere co llected from the blood of healthy volunteers. Blood was allow ed to
clot overnight a t 5°C and subsequently cen trifu g ed a t 500 x g. Sera was
14
co llected and sto red at -20°C until use. R ep eated freezin g and thaw ing, up to
six tim es, had no e ffe c t on 2D gel m igration p a tte rn s.
Double Im m unodiffusion. Double im m unodiffusion was perform ed in 1%
(w/v) agarose containing 0.5 M T ris-H C l, pH 7.5, and 0.02% sodium azide to
prevent m icrobial grow th. Im m unodiffusion p la te s w ere 45 mm x 95 m m .
Wells of 4 mm d iam eter w ere punched into th e agarose and sam ples up to 20
} x l w ere placed in each w ell. D iffusion proceeded overnight a t 5°C . P recip itin
arcs w ere visualized and photographed on a viewing box w ith an oblique light
source.
Two dim ensional iso electric focusing SD S-polyacrylam ide gel electropho
resis. P ro tein sam ples w ere resolved by two dim ensional iso elec tric focusing
SD S-polyacrylam ide gel electrophoresis (2D gels). Iso electric focusing was the
first dim ension, follow ed by sodium dodecyl sulf at e-polyacrylam ide gel
electrophoresis of the focused com ponents in the second dim ension (O’ F arrell,
1975; Anderson and Anderson, 1977). F irst dim ension gels w ere tubular, 12
mm len g th and 3 mm d iam eter, com posed of 4.0% polyacrylam ide (3.8%
acrylam ide, 0.2% BIS), 9 M urea, 2% NP-40, and 2% am pholines. E ither 0.4%
rib o flav in -5 -p h o sp h ate (R5P) or 0.01% am m onium p ersu lfate (APS) was used
as in itia to r of polym erization. TEMED, 0.03% or 0.05%, was th e c ata ly st w ith
R 5P or APS, resp ectiv ely . G radients of pH 4 to 6 or pH 5 to 7 w ere m ade by
m ixing a 4:1 ra tio of Ampholines pH 4-6 or pH 5-7 w ith Ampholines pH 3.5-10.
G radient of pH 3.5 to 5 was c re ate d by a 3.2 : 1.8 : 1 ra tio of Am pholines pH
2.5-4, pH 3.5-5, and pH 4-6 resp ectiv ely . G radient of pH 3.5 to 10 used only
am pholines pH 3.5-10. Samples containing up to 1500 \ig p ro tein w ere
15
com bined w ith an equal volum e of sam ple denaturing solution consisting of 2%
SDS, 5% 8 -m ercaptoethanol, and 10% glycerol. Only SDS o f th e highest p u rity
was found to be suitable. Low er p u rity SDS causes a rtifa c ts on 2D gels.
The sam ple m ixture was h e ate d 3 m inutes a t 100°C p rio r to iso electric
focusing. D enatured sam ple w as p laced atop a first dim ension gel, then
overlaid w ith a 2% Ampholine solution of the sam e pH range. The rem aining
tube volume was filled w ith cath o ly te and voltage was applied. A nolyte was
0.01 M H^PO^ and cath o ly te was 0.02 M NaOH (degassed). Gels w ere not
prefocused prior to sam ple application. Iso electric focusing was perform ed
w ith sam ple m igration tow ards the anode for 6,000 to 7,000 volt-hours,
overnight, a t constant voltage. The last hour of focusing was perfo rm ed a t
800 volts. Tap w ater c ircu lated through the cooling ja c k e t of the anolyte
cham ber to p rev en t overheating. A fte r focusing was com plete, gels w ere
extruded by rim m ing th e tubes w ith a needle and syringe filled w ith deionized
w ater. The pH g rad ien ts w ere read on the surface of the gels using a m icro
com bination pH probe or by slicing the gel into 1 cm sections, soaking each
section overnight in distilled w ater and subsequently reading solution pH
values. The p i values o f p ro tein s w ere determ ined from linear regression plots
of gel length vs. pH. The pH m easurem ents w ere made a t 21°C.
Second dim ension slab gels w ere 1.5 mm thick, 10% polyacrylam ide
(9*7% acrylam ide, 0.3% BIS) containing 0.375 M Tris-H Cl, pH 8.8, 0.1% SDS
and 5% glycerol. G lycerol was used to c re a te a sharp upper surface when gel
is cast. Stacking gels w ere 4.5% polyacrylam ide (4.4% acrylam ide, 0.1% BIS),
0.125 M Tris-HCl, pH 6.8, 0.1% SDS. In stacking and running gels, 0.033%
am m onium p ersu lfate and 0.05% TEMED w ere used as in itia to r and cataly st,
respectively.
16
F irst dim ension gels w ere e ith er stored frozen a t -70°C or used im m edi
a tely for electrophoresis in the second dim ension. A tube gel was placed on
top of a stacking gel and w arm (60°C) 1% agarose solution, containing 0.125 M
Tris-H Cl, pH 6.8, and 0.1% SDS, was poured over the first dim ension gel.
When solidified, the agarose held th e gel firm ly in place. A sm all well was
punched in the solid agarose n ear one end o f the tube gel. Five m icro liters of
a solution of m olecular w eight standards was placed in th e well and covered
w ith warm agarose. The m olecular w eight standards, which w ere soluabilized
in sam ple denaturing solution and h eated for 3 m inutes a t 100°C, w ere as
follows: carbonic anhydrase, ovalbum in, bovine album in, phosphorylase b, 3”
galactosidase and myosin, re c o n stitu ted as described by the m an u factu rer
(Sigma). In some cases, BioRad low m olecular w eight standards w ere used,
which contained lysozym e and soybean trypsin inhibitor b u t o m itted myosin
and 3 “galactosidase.
For the second dim ension gels, a running b u ffer of 2% sodium dodecyl
sulfate, 0.025 M Tris and 0.192 M glycine was used for the first tw enty
m inutes of electrophoresis. This was replaced by a sim ilar b u ffer consisting of
the sam e m olarities of Tris and glycine, b u t containing 0.1% SDS, and several
drops of 0.1% brom ophenol blue solution added as tracking dye. For the first
80 m inutes, electrophoresis proceeded a t a co n stan t cu rren t o f 20 m illiam ps.
The rem ainder of electrophoresis was com pleted a t 40 m illiam ps p e r gel u n til
the tracking dye reached the gel b o tto m . Cold (5°C) w ater was circu lated
through the cooling cham ber to p rev en t overheating.
Staining o f polyacrylam ide gels for p rotein, and drying. P roteins w ere
visualized by staining slab gels overnight in a solution o f 0.025% Coom assie
17
B rilliant Blue R/25% isopropanol/10% a ce tic acid. D estaining was in sev eral
changes of a 10% isopropanol/10% a ce tic acid solution. Gels w ere gently
a g ita te d on a ro ta ry shaker. D estained gels w ere dried on cellulose sh eets
using a h eated slab gel dryer a tta c h e d to a high vacuum pump.
Staining of polyacrylam ide gels for glycoproteins. G lycoproteins w ere
visualized on polyacrylam ide gels by th e periodic acid-Schiff's reag en t staining
procedure (PAS reaction) described by Fairbanks, Steck, and W allach (1971).
Gels w ere fixed for several hours in each o f the following fixing solutions: a)
25% isopropanol/10% acetic acid, b) 10% isopropanol/10% a ce tic acid, c) 10%
a ce tic acid. Gels w ere stained by soaking in a) 0.5% (w/v) periodic acid for 2
hours, b) 0.5% (w/v) sodium arzen ite/5 % acetic acid for 1 hour, c) 0.1% (w/v)
sodium arzen ite/5 % a ce tic acid, for 3 w ashes o f 20 m inutes duration, d) 10%
a ce tic acid for 20 m inutes, e) S chiff’ s reag en t overnight, in th e dark. D estain
ing was perform ed in the dark by several changes of 0.1% (w/v) sodium
m etab isu lfite dissolved in 0.01 HC1. G lycoproteins w ere visualized as pink
spots.
Silver-staining m ethods. P olyacrylam ide gels containing sm all am ounts
of p ro tein s and glycoproteins w ere stained using e ith e r of tw o silver staining
procedures. The first, outlined by Wray e t al. (1981) is sensitive for p ro tein s
and relativ ely insensitive for glycoproteins. Gels w ere fixed by one overnight
wash and two additional 1 hour w ashes in 50% (v/v) m ethanol. Staining was for
15 m inutes in 8 m g/m l silver n itra te solution containing 0.8% (v/v) am m onium
hydroxide and 0.08% (w/v) sodium hydroxide. A fter a short wash in deionized
w ater, a fresh solution of developer, 0.02% (v/v) form aldehyde and 0.005%
18
(w/v) citric acid in w ater, was added to th e gels. A fter approxim ately 10
m inutes, brown spots appeared. D evelopm ent was stopped by addition of
Kodak Rapid Fix, followed by a rinse in H ypo-C lear to rem ove fixer. A fter
additional washes in 25% m ethanol, gels w ere dried.
The second silver staining m ethod is m ore sensitive for glycoproteins
than for non-glycosylated p ro tein s (Dubray and B ezard, 1982). Fixing was
p erform ed overnight in 25% (v/v) isopropanol/10% a ce tic acid, followed by
th ree washes in 7.5% a ce tic acid. The gels w ere then soaked in 0.2% (w/v)
aqueous periodic acid for one hour a t 5°C . This was followed by six w ashes in
deionized w ater. The rem aining procedure follows th a t described in the first
m ethod, above.
Fluorography and autoradiography. Two dim ensional gels containing
35 14
( S)- or ( C )-labeled com ponents w ere tre a te d w ith EnHance, a fluoro
graphic enhancing solution. Gels w ere soaked in this solution for one hour,
washed for one hour in d istilled w ater which p re c ip ita te d the enhancer, then
125
dried. Gels containing ( I)-labeled pro tein s w ere dried w ithout tre a tm e n t
w ith EnHance. Dry gels w ere exposed to X -ray film in X -ray exposure holders
a t -70°C for varying periods of tim e. Gels containing (^ ^ 1 )-labeled com po
nents w ere occasionally autoradiographed w ith a Cronex intensifying screen.
Exposed film was processed as recom m ended by the m anufacturer.
E lectrophoretic T ransfer (W estern B lotting). P roteins and glycoproteins
w ere electro p h o retically tra n sfe rre d from 2D gels onto u n tre a te d n itro cellu
lose in a TE 42 transphore apparatus. The procedure was adapted from
Towbin, Staehelin, and Gordon (1979), and B u rn ette (1981). A fter 2D gel
19
electrophoresis, gels w ere fixed overnight in tra n sfe r b u ffer consisting o f 0.15
M glycine, 0.02 M Tris-H Cl, pH 8.3, and 20% m ethanol. U n treated n itro cellu
lose sheets, M illipore type HA having 0.45 m icron pore size, w ere used. A
single gel was placed in the gel holder, or c assette , which is assem bled
according to the m an u factu rer's in structions. The 2D gel was held tig h tly
against th e nitrocellulose sh eet w ithin th e gel c assette . The c a sse tte was
subm erged in tra n sfe r b u ffer precooled to 5°C by a circulating w ater bath.
The leads w ere connected so th a t the nitrocellulose sh eet was closest to the
anode, allow ing anionic p ro tein s to m ig rate from the gel to the sheet under the
influence of th e e lec tric field. A cu rren t of 200 m illiam ps was applied for one
hour.
A fter tran sfer, blots w ere eith e r stained for p ro tein or carried through
im m unoautoradiography. Blots w ere stained by soaking for 5 m inutes in a
solution of 0.2% (w/v) Coom assie Blue/40% (v/v) m ethanol/10% (v/v) a ce tic
acid. They w ere rapidly destained in 2 washes, for 3 m inutes each, in 90%
(v/v) m ethanol/2% (v/v) a ce tic acid, then pressed dry betw een sheets of filte r
paper.
Im m unoautoradiography of W estern b lo ts. Im m unoautoradiography was
carried out by soaking W estern b lo ts in the appropriate antibody solution
125
followed by I-protein A (from Staphylococcus aureus), using a m ethod
adapted from Towbin e t al. (1979) and B u rn ette (1981). Im m ediately
following electro p h o retic tra n sfer, each b lo t was soaked for a minimum of 60
m inutes a t 37°C in 0.01 M Tris-H C l b u ffered saline, pH 7.4, (TBS) containing
3% (w/v) bovine serum album in (TBS/BSA). A fter a quick rinse in 0.01 M TBS,
the b lo t was soaked for 90 m inutes a t 37°C in TBS/BSA containing antibodies
20
a t su fficien t co n cen tratio n to bind five tim es m ore antigen than is p re sen t on
the b lo t. The b lo t was th en w ashed as follows, on a ro ta ry shaker a t room
te m p e ratu re: 20 m inutes in TBS, tw ice for 20 m inutes in TBS containing
0.05% NP-40, and 20 m inutes in TBS. The b lo t was then soaked in TBS/BSA
6 125
containing 1 x 10 cpm /m l I-P ro tein A (specific activ ity 1 |i.Ci p er Jig or
g reater) for 30 m inutes a t room tem p eratu re. The washing schedule of TBS
and TBS/NP-40 was rep eated , and the b lo t was pressed dry b etw een sh eets of
filte r paper. The b lo t was w rapped in p lastic and exposed to Kodak X -ray
film in a holder equipped w ith an intensifying screen. The film was developed
as p er m an u factu rer's instructions.
D ensitom etry. The o p tical density (OD) of stained isotype spots on some
2D gels as w ell as darkened spots on autoradiographs and fluorographs was
d eterm ined using a m anual d en sito m eter equipped w ith a 2 mm a p ertu re. The
following filters used for d en sitom etry: red filte r for Coom assie B lue-stained
spots, green filte r for periodic acid-S chiff's-stained spots, and no filte r for
*
X -ray film spots. The p e rc e n t of to ta l p ro tein p er isotype was determ ined
from op tical density readings by the form ula:
spot OD m inus background OD jqq
21 (spot OD m inus background OD)
M olecular sieve chrom atography. Sephadex G-25, G-100 and G-200 w ere
hydrated as p er m an u factu rer's instructions. Depending on sam ple type and
volum e, colum ns ranging from 10 ml to 190 ml bed volum e w ere poured.
Colum ns used in creatin e kinase experim ents w ere p reeq u ilib rated w ith 0.1 M
Tris-H Cl, pH 8.0, containing 0.1% (v/v) B -m ercaptoethanol. O ther experi-
21
m ents u tilized 0.05 M phosphate b u ffered saline, pH 7.2 (PBS), containing
0.02% (w/v) sodium azide. For radioiodinated sam ples, PBS containing 0.1%
(w/v) BSA was used. When appropriate, colum ns w ere calib rated for m olecular
w eight by applying a m ixture of aldolase (158,000 mol. wt.), bovine serum
album in (68,000), ovalbum in (43,000) and cytochrom e c (12,500). Excluded and
included volum es w ere d eterm ined using dex tran blue and phenol red, resp ec
tively. Volumes of applied sam ples w ere 1% to 5% of the colum n bed volum e.
Column flow was downward by g ravity and appropriate sam ples w ere run a t
5°C . Sample application and elution w ere perfo rm ed in th e sam e b u ffer used
for preequilibration. E luted non-radioactive p ro tein was m onitored by
absorption a t 280 nm . R adioiodinated p ro tein was m onitored by counting sm all
aliquots of each fractio n in a gam m a counter.
Ion exchange chrom atography. D iethylam inoethyl-cellulose was the
m atrix used for ion-exchange chrom atography. The m icrogranular form was
obtained presw ollen, containing 70% w ater by w eight, 1 m illiequivalent p e r
gram of dry w eight. The m atrix was precy cled by a lte rn a te washes in 0.5 N
HC1 and 0.5 N NaOH. A colum n of 75 m l bed volum e was poured and
preeq u ilib rated w ith 0.1 M Tris-H Cl, pH 8.0, containing 0.1% (v/v) $--naercap-
to ethanol (Tris-$M E buffer). Samples o f 16 to 20 m l containing approxim ately
4 mg p ro tein w ere run into the column. Non-binding m aterial w as elu ted by
washing w ith several colum n volum es of Tris-3M E b u ffer. Bound m aterial was
eluted w ith a linear gradient of increasing ionic strength, 0 to 0.5 M NaCl in
Tris~3ME b u ffer. P rotein was m onitored by absorption a t 280 nm . C onductiv
ity was d eterm ined by m easuring m illim hos of individual fractio n s w ith a
conductivity m eter.
22
R adioiodination. P ro tein s w ere radioiodinated by a m odification o f th e
chloram ine T m ethod of H unter and Greenwood (1962). Sam ples containing
from 5 to 50 jig p rotein, in volum es up to 50 |il of PBS, pH 7.2, were com bined
w ith 25 (J.1 of 0.1 M sodium phosphate buffer, pH 7.2, and 5 |J.l (0.5 mCi) of Na
125
I in a capped 1.5 ml m icro cen trifu g e tube. T w enty-five |il of C hloram ine T
(4 mg/ml) was added and quickly vortexed. A fter 1 m inute, 25 |il of sodium
m etabisulfite (12 mg/ml) and 100 p.1 of potassium iodide (10 mg/ml) in PBS
containing 1% (w/v) BSA w ere added to stop the reactio n , and the tube again
vortexed. The con ten ts of the tube w ere then p laced on a Sephadex G-25
colum n o f 10 m l bed volum e and elu ted w ith PBS containing 1% (w/v) BSA.
125
R adiolabeled p ro tein was obtained in the void volum e, and I-salt was
retain ed on th e column.
C reatin e kinase enzym atic assays. C reatin e kinase assays w ere kindly
perform ed by Dr. L aw rence Silverm an a t the H ospital of the Good Sam aritan,
Los Angeles, CA. C reatin e kinase isoenzym e id entity was established by
agarose electrophoresis in b a rb ita l b u ffer a t pH 8.6. Bands w ere visualized by
overlaying w ith specific su b strate creatin e phosphate in th e p resen ce of ADP.
This reactio n is coupled to th e production of fluorescent NADH, as outlined:
p t r
cre atin e phosphate + ADP — ■ > creatin e + ATP
MgClz
L -cysteine
ATP + glucose -
Hexokinase
glucose-6-phosphate + ADP
glucose-6-phosphate + NAD ^
G-6PDH
■ * > 6-phosphogluconate + NADH
23
Bands of activ e enzym e fluoresce and are d e te c te d a t 365 nm (E levitch, 1973;
Silverm an e t al. 1978).
T o tal c re atin e kinase activ ity was determ ined using a m odified Rosalki
procedure (Rosalki, 1967) w here NAD is used in stead of NADP to reduce non
specific activ ity . Sim ilarly, this assay is coupled to th e production of NADH,
which is k in etically m onitored a t 340 nm.
C reatin e Kinase BB p u rificatio n from p ro sta tic fluid. P ro sta tic fluid was
used as a source o f c re atin e kinase-BB (CK-BB). P u rificatio n procedure was a
m odification of th e m ethod of A rm strong, Lowden and Sherwin (1977). P ros
ta tic fluid was cen trifu g ed a t 49,000 x g and 2.5 m l of supernant was applied
to a Sephadex G-100 column having 190 ml bed volum e. Elution was
perfo rm ed w ith 0.1 M Tris-H Cl, pH 8.0, containing 0.014 M 8 -m ercap to eth an o l
(0.1% v/v), and fractio n s w ere te ste d for CK activ ity . A ctive fractio n s w ere
pooled and applied to a D EA E-cellulose colum n, using the sam e b u ffer. This
colum n was eluted w ith a linear g rad ien t of 0 to 0.5 M N aCl in T ris-$ -
m ercap to eth an o l b u ffer. F ractions w ere again assayed for CK a ctiv ity and the
a ctiv e fractio n s w ere pooled.
O rgan c u ltu re . Benign p ro sta te hyperplasia (BPH) specim ens w ere deliv
ered under sterile conditions d irectly from the operating room . A djacent
tissue was processed for routine pathology and the diagnosis o f BPH confirm ed
3
in each case. Tissue was cu t into cubes o f approxim ately 1 mm and placed
atop sterile trian g u lar screen s m easuring 20 mm p e r side. Two to th re e loaded
screens w ere p laced in a 60 mm x 15 mm ste rile p lastic p e tri dish and 3 m l of
cu ltu re m edium was added, su fficien t to m aintain th e tissue explants a t the
24
air-liquid in te rfa c e. The m edium used for (*^C)-glucosam ine labeling was
Medium 199 containing glucose (1 m g/m l), penicillin (100 units/m l), stre p to -
14
m ycin (100 p.g/ml), 0.5% to 2.5% (v/v) fe ta l bovine serum and 5 j-lCi of ( C)-
glucosam ine per dish. Explants w ere incubated for 48 hours in the presence of
rad io activ e precursor a t 37°C in a w a te r-sa tu ra te d atm osphere of 95% room
air-5% CO^. In som e cases this m edium was replaced a fte r 48 hours w ith
fresh m edium minus rad io activ e p recursor, and incubation was continued for
another 48 hours. The spent m edium rem oved from p e tri dishes was
centrifuged for 10 m inutes a t 1,000 x g to rem ove debris, and th e supernate
was exhaustively dialyzed against 0.01 M PBS, pH 7.2, containing 0.02% (w/v)
sodium azide a t 5°C.
PC-3 cell culture. P ro sta te carcinom a cell line PC-3 is an epitheloid
cell line established from a poorly d ifferen tia te d hum an p ro sta te adenocar
cinom a which had m etastasized to bone (Kaighn e t al., 1979). C ells w ere
m aintained a t 37°C in a w a ter sa tu ra te d atm osphere of 95% air/5% CO^. The
grow th medium RPMI-1640 was supplem ented w ith 10% fe ta l bovine serum ,
insulin (10 |!g/m l), fungizone (5 |J,g/ml), pencillin (50 units/m l) and stre p to
m ycin (50 |ig/m l). In som e in stan ces fungizone was replaced w ith gentam ycin
(10 |ig/m l).
M etabolic labeling of cellular glycoproteins was perform ed in the sam e
14
medium containing 0.1% to 2% fe ta l bovine serum and 5 |iC i of ( C)-
2
glucosam ine. One m illion PC-3 cells, p lated in 175 cm tissue cu ltu re flasks,
w ere grown until m id d le-to -late-lo g arith m ic grow th phase was reached and
approxim ately five m illion cells w ere p resen t. Fresh m edium containing
14
( C)-glucosam ine was added and incubation continued for 24 to 48 hours. A t
____________________________________________________________________________25_
th a t point the m edium was co llected , cen trifu g ed a t 500 x g to rem ove debris,
and extensively dialyzed a t 5°C against 0.01 M PBS, pH 7.2, containing 0.02%
sodium azide to p rev en t m icrobial grow th. D ialyzed medium was eith er
lyopholized or stored a t -20°C .
M etabolic labeling of p ro tein s was also perform ed by growing cells in the
35
p resence of ( S)-m ethionine. Nine m illion cells p lated in a tissue cu lture
flask w ere labeled for 24 hours in 15 ml of m eth ionine-free RPMI-1640
35
medium containing 150 [iCi o f ( S)-m ethionine, 100 U /m l penicillin, 100
{J-g/ml streptom ycin, 50 [ig/p.1 gentam ycin, and 3% fe ta l bovine serum . Spent
medium was co llected , cen trifu g ed a t 500 x g, and dialyzed extensively against
PBS a t 5°C.
Tissue hom ogenization. Benign p ro sta te hyperplasia tissue was hom oge
nized on ice using a Brinkm an P olytron hom ogenizer equipped w ith a 1 cm tip.
Several gram s o f tissue was com bined w ith 3 tim es its volum e of 0.05 M PBS,
-4
pH 7.2, containing 1 x 10 M phenylm ethylsulfonylfluoride, a serine pro tease
inhibitor. Tissue was thoroughly hom ogenized for 3 m inutes, followed by a
cen trifu g atio n a t 600 x g, 4°C , 15 m inutes, to rem ove large p ieces o f debris.
The sup ern atan t from this step was recen trifu g ed a t 31,000 x g for 60 m inutes,
4°C . The resulting su p ern atan t was stored a t 5°C u n til fu rth er use.
P ro tein d eterm in atio n . P ro tein co n cen tratio n s w ere determ ined using
the BioRad p ro tein assay. This is based on the B radford (1976) assay in which
absorbance m aximum of an acidic solution of Coom assie B rilliant Blue G-250
shifts from 465 nm to 595 nm when the dye binds p ro tein . Bovine gam m a
globulin was used as a standard.
26
P erch lo ric acid p recip itatio n . Serum sam ples and cell cu ltu re superna
ta n ts w ere p re c ip ita te d a t 0°C using ice cold 0.6 _ M perchloric acid. Equal
volum es of acid and sam ple w ere m ixed w ith dropw ise addition of acid. The
sam ples w ere set on ice for one hour, followed by cen trifu g atio n at 4°C , 9,000
x g, 15 m inutes. G lycoprotein-enriched su p ern atan ts w ere n eu tralized w ith ice
cold 1.2 N KOH and salt p re c ip ita te s w ere p elleted at 9,000 x g. The volum e
of sup ern atan ts was reduced by n eg ative pressure co n cen tratio n or u ltra filtra
tion.
T rich lo ro acetic acid p recip itatio n . T rich lo ro acetic acid (TCA) p re c ip ita
tions of radiolabeled proteins w ere p erfo rm ed by m ixing equal am ounts of cold
(5°C) 20% (w/v) TCA and sam ple. Five to 10 p.1 of hum an serum was added to
th e m ixture as a source of p recip itab le, unlabeled p rotein. The p re c ip ita te s
w ere allow ed to form for a minimum of 2 hours a t 5°C , then w ere p elleted by
15 m inutes of cen trifu g atio n a t 2,500 x g. P ellets w ere washed tw ice in the
sam e volum e of 10% TCA by thorough vortexing and cen trifu g atio n . Samples
125
labeled w ith I w ere placed d irectly in a gam m a counter to determ ine cpm .
35 14
Those labeled w ith ( S) or ( C) w ere solubilized in 100 to 300 ( J L l of Protosol
and then com bined w ith Econofluor. R ad io activ ity levels w ere d eterm ined in a
scin tillatio n counter.
P hosphotungstic a cid /tric h lo ro a ce tic acid p recip itatio n . To p re c ip ita te
radiolabeled glycoproteins and p ro tein s m ore efficien tly , a volum e of radio-
labeled sam ple was m ixed w ith an equal volum e of cold 2% (w/v) phosphotung
stic acid (PTA)/20% (w/v) TCA solution. The resulting p re c ip ita te was washed
w ith a 1% PTA/10% TCA solution as outlined above for TCA p recip itatio n .
27
A cetone p recip itatio n . Sodium dodecyl su lfate was rem oved from
p ro tein solutions (Hager and Burgess, 1980) by m ixing 1 volum e of p ro tein
solution w ith 4 volum es of ice cold aceto n e. The p re c ip ita te form ed for 30
m inutes in a dry ice-aceto n e bath. P re c ip ita te was p elleted at 4°C by a 10
m inute cen trifu g atio n at 14,500 x g. The p e lle t was resuspended in 2% (w/v)
SDS/5% (v/v) £-m ercaptoethanol/10% (v/v) glycerol, and subjected to tw o
dim ensional gel electrophoresis.
Im m unopr ecipi ta t ions. R abbit antibodies w ere used in a tte m p ts to
im m u noprecipitate th e following hum an serum com ponents from various sam
ples, including radiolabeled cell and organ cultures: a - l- a c id glycoprotein, a -
1-antichym otrypsin, a -l-p ro te in a s e inhibitor and haptoglobin. Imm une com
plexes w ere p rec ip ita ted in d irectly w ith eith er goat a n ti-ra b b it im m unoglob
ulin (GARGG) or form alin-fixed Staphylococcus aureus (Staph A) cells
(K essler, 1976; Cullen and Schw artz, 1976). Fixed cells of Staph A Cowan I
strain contain p ro tein A in the cell walls. This p ro tein binds to th e Fc portion
of rab b it IgG m olecules and has g re a te r affin ity for im m une com plexes than
for free IgG (Kessler, 1975). The presence of excess Staph A cells allows rapid
p recip itatio n of im m une com plexes by low speed centrifu g atio n , elim inating
the need for second antibody titra tio n .
Sam ples containing from 1.0 x 10^ to 3.9 x 10^ dpm of (^ C )-la b e le d
3
com ponents w ere used. R adioiodinated sam ples contained 8.0 x 10 to 4.0 x
10^ cpm . All sam ples and p ro tein solutions w ere centrifuged a t high speed
p rior to p recip itatio n . E ither PBS, pH 7.2, or TBS, pH 7.4 containing 0.05%
NP-40, w ere used as b u ffers. D epending on sam ple size, polypropylene or glass
tubes of 1.5 ml to 50 m l size w ere used. E xtraneous p ro tein , such as 1% (w/v)
28
solution of BSA in PBS, was added to m ost experim ents to reduce non-specific
binding of radiolabeled com ponents to tubes. A ppropriate am ounts o f rabbit
antibody solutions w ere placed in te s t tubes, and equivalent am ounts o f non-
im m une rabbit serum w ere placed in co n tro l tubes. Samples w ere added and
im m une com plexes w ere allow ed to form overnight a t 5°C, with ag itation.
The following day a 10% (w/v) suspension of Staph A cells was added to the
tubes and incubated for two hours. Ten (J.1 of 10% Staph A suspension was
su fficien t to bring down com plexes form ed by 1 |-ll of im m une serum . Staph A
cells w ere p elleted by cen trifu g atio n a t 3,000 x g for 15 m inutes, 5°C . P ellets
w ere w ashed tw ice in b u ffer and re p e lleted a fte r each wash. Final p e lle ts
w ere e x tra c te d w ith 6 M u re a containing 2% (w/v) SDS, a t 100°C for 3 m inutes
(K essler, 1975). E x trac te d (^ C )-co m p o n en ts w ere com bined w ith PCS-IT
scin tillatio n co ck tail and counted in a scin tillatio n counter. C om ponents
124
containing I w ere counted d irectly in a gam m a counter, w ithout ex tractio n .
For som e im m unoprecipitations, GARGG w as used in p lace of Staph A
cells. A fter addition of su fficien t GARGG to p re c ip ita te the rab b it Ig, tubes
w ere incubated for 5 to 6 hours a t room te m p eratu re. P re c ip ita te s w ere
p elleted by cen trifu g atio n a t 5,000 x g, washed tw ice w ith b u ffer, and
125
rep elleted . For d etectio n of I, p ellets w ere d irectly counted in a gam m a
14
counter. W ith ( C )-com ponents, the p e lle t was solubilized to 200-500 |J.l of
Protosol, com bined w ith Econofluor, and counted in a scin tillatio n counter.
For 2D gels, the p e lle ts w ere solubilized in 2% SDS/5% $ -m ercap -
toethanol/10% glycerol, h eated and placed on first dim ension gels.
Im m unoaffinity chrom atography. In tw o sep arate experim ents, antibody
or antigen w ere coupled to Sepharose 4B to c re a te im m unoaffinity colum ns.
29
For an antibody-coupled im m unoaffinity m atrix , 9 mg of rab b it an ti
hum an a -l-a c id glycoprotein im m unoglobulins w ere coupled to 1 gram of
cyanogen brom ide (CNBr) activ ated -S ep h aro se 4B using th e p ro to co l recom
m ended by m an u factu rer. The C N Br-Sepharose 4B was h y d rated in 1 mM HC1.
A ntibody solution was dialyzed against coupling b u ffer: 0.1 M NaCO^, pH 8.3,
containing 0.5 M N aCl. The Sepharose gel was suspended in coupling b u ffer
and m ixed w ith antibody solution in a capped te s t tube by rocking overnight a t
5°C . The rem aining activ e groups of the gel w ere blocked by washing the gel
in 1 M ethanolam ine for 16 hr a t 5°C . The coupled gel was w ashed a lte rn a te ly
w ith high and low pH b u ffer solutions. Coupling b u ffer was used as high pH
solution and 0.1 M sodium a c e ta te , pH 4.0, containing 0.5 M N aCl was used as
low pH b u ffer.
The gel was tra n sfe rre d to PBS, pH 7.2 and poured into a disposable 5 ml
syringe plugged w ith glass wool. The colum n w as fu rth e r w ashed w ith several
bed volum es of PBS. Sam ples of 0.5 m l or less w ere allow ed to e n ter the
colum n, flow was stopped, and a tw o hour incubation period followed.
Subsequently, unbound m a te ria l was washed away w ith five bed volum es of
PBS. Bound com ponents w ere elu ted w ith e ith e r 0.1 M glycine containing 0.15
M N aCl adjusted to pH 2.5 w ith HC1, or 3 M potassium th io cy an ate in 0.15 M
N aCl, pH 2.5. The pH of elu ted fractio n s was im m ediately adjusted to pH 8
w ith solid Tris to m inim ize p ro tein dam age. Eluted m a te ria l was m onitored
for p ro tein by absorption a t 280 nm. For rad io activ e sam ples, an aliquot of
each frac tio n was used to determ in e dpm in a scin tillatio n counter.
A ntigen im m obilized to Sepharose 4B was used to purify antibodies from
im m une rab b it serum . a -l-A c id glycoprotein, p u rified from hum an serum ,
was coupled to C N B r-activated Sepharose 4B as outlined above, except 0.1 M
30
sodium b o rate, pH 8.3, containing 0.5 M NaCl was the high pH washing b u ffer.
Colum n was equilibrated w ith PBS. R abbit antiserum raised against a - l- a c id
glycoprotein was h eated a t 56°C, 30 m inutes, to d e ac tiv a te com plem ent.
S atu rated am m onium su lfate was added to bring th e solution to 40%, the
p re c ip ita te was cen trifu g ed and the p e lle t dissolved in PBS. This solution was
dialyzed extensively against PBS to rem ove ammonium su lfate. The dialyzed
Ig frac tio n was applied to the colum n, incubated and unbound p ro tein washed
away as described above. Bound p ro tein was eluted w ith 0.1 M glycine-H Cl,
pH 3.5, containing 3 M potassium th io cy an ate. F ractio n s w ere analyzed as
above.
R abbit Im m unization. A 4-w eek old New Zealand w hite rab b it was
im m unized against a -l-a c id glycoprotein from hum an serum . The rab b it was
in jected in trad erm ally a t 2 w eek in terv als w ith 60 to 100 |J,g of a -l-a c id
glycoprotein in 0.5 ml PBS m ixed w ith 0.5 ml of Freund's com plete adjuvant.
The last injection was w ithout any adjuvant. Blood was co llected by cardiac
puncture ten days a fte r th e la st injection and was allow ed to clot overnight a t
5°C. Serum was co llected by cen trifu g atio n a t 500 x g and sto red a t -2 0 °C .
U ltra filtra tio n . C o n cen tratio n of som e sam ples was perform ed by u ltra
filtra tio n in A m icon stirre d cells. Depending on sam ple volum e, cell models
having volum es of 3, 10 or 50 m l w ere used. D iaflo u ltra filte r m em branes of
e ith er type PM or YM w ere em ployed, the la tte r type dem onstrating low non
specific binding of p roteins. M olecular w eight cu to ffs for th ese m em branes
w ere eith er 10,000 or 30,000. The pressure of in ert N^ gas used for
u ltra filtra tio n was b etw een 25 and 40 pounds p er square inch.
31
N egative pressure co n cen tratio n and dialysis. Some sam ples w ere
co n cen trated by negative p ressure co n cen tratio n w ith co n current dialysis. A
m odel 320 Micro ProDiCon neg ativ e pressure m icroprotein dialysis co n cen tra
to r was used. In this system , th e d ilu te sam ple is confined w ithin a v e rtic a l
dialysis m em brane which is im m ersed in a continuously stirre d d ialysate, under
vacuum . The dialysate used was PBS, pH 7.2, containing 0.02% (w/v) sodium
azide to prevent m icrobial grow th. C o n centration was p erfo rm ed a t 5°C,
using a dialysis m em brane having a m olecular w eight c u to ff of 10,000 daltons.
C o n cen trated sam ple was co llected in a reserv o ir a t the bottom of the v e rtic al
m em brane.
q -l-A c id glycoprotein (AGP) co n cen tratio n in serum . The am ount of
to ta l AGP p er ml of norm al serum was determ ined by ra te nephelom etry using
the Beckm an Im m unochem istry System (Beckman Instrum ents, Inc., Fullerton,
CA).
P u rificatio n of q -l- a c id glycoprotein from norm al hum an serum . Human
blood donated by a healthy v o lunteer was used as a source of q -l- a c id
glycoprotein (AGP). Blood w as draw n into V acutainer ev acuated tubes w ithout
anticoagulants and was allow ed to clo t overnight a t 5°C. Serum was co llected
by cen trifu g atio n a t 1,000 x g. The bulk of p ro tein s in 16 ml of serum was
rem oved by p recip itatio n in 0.3 N p erchloric acid on ice for 60 m inutes.
P re c ip ita te was p elleted by cen trifu g atio n a t 9>000 x g, 5°C , and the superna
ta n t was n eu tralized w ith ice cold 1.2 N KOH. R esulting salt p re c ip ita te was
rem oved by cen trifu g atio n as above. This su p ernatant was co n cen trated to 0.7
ml using an Amicon Model 12 u ltra filtra tio n ap paratus having 10 ml volum e
32
and equipped w ith a PM 10 m em brane. The c o n ce n tra te was applied to a
Sephadex G-100 colum n w ith bed volum e of 170 ml, and eluted w ith 0.05 M [
PBS, pH 7.2. Eluted fractio n s w ere m onitored for p ro tein s by absorbance at
280 nm. P ro tein peaks w ere te ste d for p resen ce of AGP by tw o dim ensional
gel electrophoresis.
N euram inidase tre a tm e n t of a -1 -a c id glycoprotein. T hirty m icrogram s
of a - l- a c id glycoprotein, p artially purified by perch lo ric acid tre a tm e n t of
hum an serum , was com bined w ith 0.3 units of C lostridium p erfringens neu ra
m inidase in 0.1 _ M sodium a c e ta te b u ffer a t pH 5.0. (One unit is defined as th e
am ount of enzym e th a t will lib e ra te 1.0 Jimole of N -acety l neuram inic acid
p er m inute a t pH 5.0 and 37°C.) This solution was incubated for 3 hrs a t 37°C
and designated as once treated -A G P . A fter this incubation, an aliquot was
rem oved and an additional 0.3 units of neuram inidase was added to th e
rem aining solution, now designated as tw ice treated -A G P . This was incubated
another 3 hrs a t 37°C . Subsequently, portions of both once tre a te d - and tw ice
treated -A G P w ere resolved on 2D gels and visualized by C oom assie Blue
staining.
T ryptic p ep tid e m aps. M icrogram q u a n titie s of a - l- a c id glycoprotein
isotypes sep arated by 2D gel electrophoresis w ere analyzed by try p tic p ep tid e
m apping using tw o-dim ensional thin layer electrophoresis-chrom atography
(Elder et al., 1977a). The isotypes w ere radioiodinated and cleaved w ith
trypsin while still in the polyacrylam ide gels. 2D gels w ere stained w ith
C oom assie Blue and destained as described above. Isotypes w ere cu t from th e
gel by scalpel. Each gel slice was placed in an individual siliconized 10 x 75
33
mm glass tube, se t on a rocking p latfo rm . Slices w ere washed four tim es in
25% isopropanol, four tim es in 10% m ethanol, and dried under an in frared
lam p.
To radioiodinate th e p ro te in w ithin a gel slice, th e following w ere
com bined in a clean tube: dried g el slice, 20 jll 0.5 M sodium phosphate (pH
125
7.5), 300 to 500 pC i o f Na ( I) in 5 |-L l volum e, and 5 jJ.1 of freshly prep ared
chloram ine T (1 m g/m l). This m ixture was incubated 1 hour a t room
te m p e ratu re. One ml o f sodium m etab isu lfite (1 mg/ml) was added and th e
m ixture incubated for 15 m inutes to stop the re a ctio n . The gel slice was
125
extensively washed in 10% m ethanol to rem ove free I, and dried under an
in frared lam p.
R adioiodinated p ro tein w ithin th e gel slice w as trypsinized by com bining
th e slice w ith 0.5 m l of trypsin, 13 to 15 u n its/m l, in 0.05 M am m onium
b icarb o n ate b u ffer, pH 8.0. Incubation was overnight a t 37°C , allowing
su fficien t tim e for enzym e and p ep tid e diffusion through the gel. Subsequent
ly, th e solution containing p ep tid es w as sh ell-frozen and lyopholized.
T ryptic p ep tid es w ere sep arated by tw o dim ensional thin layer e le c tro
phoresis -chrom atography. The m atrix was cellulose, 160 m icrons thick on
p lastic backing, w ithout binder or fluor, cut to 10 x 10 cm size. F irst
dim ension was thin layer electrophoresis, run on a fla t bed electro p h o resis
appratus. B uffer I for this dim ension w as a c e tic acid: form ic acid: d istilled
w ater, 15:5: 80. Lyopholyzed sam ple from a single g el slice w as dissolved in
5
20 (J.1 b u ffer I. T hree j-ll, containing approxim ately 5 x 1 0 cpm, was sp o tted
onto one corner of a cellulose p la te , which w as subsequently w etted w ith a
fine spray o f b u ffer I. The p la te w as placed on the cooling block of the fla t
bed u n it, k ep t a t 5°C by a circu latin g w ater b ath . R eservoirs filled w ith
34
b u ffer I w ere connected to th e p la te by w icks m ade of W hatm an 3 MM
chrom atography pap er. E lectrophoresis was for 15 m inutes a t 1000 volts,
constant voltage. The p la te was subsequently rem oved and air dried.
P eptides sep arated in th e first dim ension w ere fu rth e r resolved in the
second dim ension by ascending chrom atography. B uffer II for this dim ension
was butanol: pyridine: a c e tic acid: distilled w ater, 32.5 : 25 : 5 : 20. P lates
w ere developed in a chrom atography tan k filled to a dep th of 2 to 3 mm w ith
b u ffer E L Tank atm osphere was kept sa tu ra te d by a sheet of filte r paper,
w etted in b u ffer II, a tta c h e d on one tank w all. U pward developm ent continued
until th e b u ffer fro n t was w ithin 5 mm of the p la te top. The p la te was
rem oved, a ir dried, and exposed to X -ray film a t -70°C . Film was processed
as p er m an u factu rer's in structions.
In d irect im m unoperoxidase staining of paraffin-em bedded tissue sec
tions. A ntigen was d e te c te d in sections of form alin-fixed, paraffin-em bedded
tissues using th e peroxidase anti-peroxidase staining m ethod of Taylor (Taylor,
1978; N aritoku and Taylor, 1981). This m ethod u tilizes th re e reag en t layers;
p rim ary antibody, bridging antibody and peroxidase-antiperoxidase com plex.
When su b strate diam inobenzidine is added, a brown p re c ip ita te form s, allowing
antigen lo calizatio n w ithin the section.
Form alin fixation and paraffin-em bedding of p ro sta te tissue was p e r
form ed by th e Surgical Pathology D ep artm en t of LAC/USC M edical C en ter.
Fixed tissue was cut into sections of 4-5 m icron thickness. The sections,
placed on glass slides, w ere h e ate d overnight at 58-59°C to m elt the p araffin
and fix the sections to the slides. These step s w ere kindly perfo rm ed in the
lab o rato ry of D r. C live R. Taylor, D ep artm en t of Pathology, USC School of
35
M edicine. A diam ond scrib er w as used to etch a circle around sections to aid
in retaining solutions.
S ections w ere dew axed and brought to alcohol by a 5 m inute wash in
xylene, tw o 3 m inute w ashes in absolute eth an o l and tw o 3 m inute w ashes in
95% ethanol, 5% H^O. The slides w ere tre a te d for 20 m inutes w ith 0.6%
hydrogen peroxide in m ethanol to rem ove endogenous peroxidase a ctiv ity ,
followed by a rinse in and a 10 m inute wash in PBS, pH 7.2.
All incubations w ere carried out in a hum idity cham ber a t room
te m p e ratu re. Slides w ere incubated for 30 m inutes w ith 1:20 dilution of
norm al swine serum in PBS, to decrease nonspecific background staining,
followed by a quick wash in PBS. Various dilutions of th e p rim ary antibody,
rab b it anti-hum an a - l- a c id glycoprotein, w ere then added to the slides and
incubated for 30-60 m inutes, followed by a 10 m inute wash in PBS. A 1:60
dilution o f bridging antibody, swine a n ti-ra b b it im m unoglobulins, was added
and incubated for 30 m inutes, followed by an o th er wash in PBS for 10 m inutes.
This procedure was re p e ate d w ith a 1:200 dilution of rab b it-P A P com plex, 30
m inute incubation. L astly, th e slides are tre a te d w ith the su b strate diam ino-
benzidine, 0.6 m g/m l in PBS containing 0.03% hydrogen peroxide, for 3-5
m inutes. Slides w ere rinsed w ell in tap w ater, co u n terstain ed w ith h em atoxy
lin, and dehydrated by reversing the w ashes in 95% ethanol, absolute ethanol,
and xylene. C overslips w ere m ounted to th e slides using Perm ount. C ontrols
w ere om ission of prim ary antibody and rep lacem en t w ith non-im m une rabbit
serum .
G lassw are siliconization. G lassw are w as siliconized by filling w ith a
solution o f 5% (v/v) dim ethyldichlorosilane in toluene, for 10 to 15 m inutes.
36
A fter this solution w as decan ted , the glassw are was rinsed w ith acetone,
washed w ith soap, rinsed w ith w ater and air dried.
Photography. Stained 2D gels, autoradiographs and fluorographs w ere
photographed using a Polaroid M P-4 Land C am era m ounted on a copy stand.
In stan t black and w hite p rin ts and n eg ativ es w ere obtained using Polaroid Type
665 P o sitive/N egative film . Gels and film s w ere set on a light box which
provided illum ination from below . Gels stained w ith Coom assie Blue w ere
photographed through a red no. 25 W ratten g elatin filter, and gels stained by
PAS re a c tio n w ere photographed through a green no. 55 W ratten filter. No
filters w ere used to photograph fluorographs and autoradiographs.
37
CHAPTER H
ANALYSIS OF PROTEINS AND GLYCOPROTEINS IN PROSTATIC FLUID
USING TWO DIMENSIONAL GEL ELECTROPHORESIS, WESTERN BLOTS,
AND IM MU NO AU TO R AD IOGR APH Y.
38
INTRODUCTION
P ro sta tic fluid contains a com plex m ixture of p ro tein s, glycoproteins,
lipids, enzym es, and polyam ines (G rayhack and Lee, 1981). This c h ap ter
discusses the tw o dim ensional electro p h o retic sep aratio n of the m acro m ole
cules found in p ro sta tic fluid. The flhid sam ples w ere obtained from re sec te d ,
hyperplastic p ro sta te tissue. Two dim ensional gel electro p h o resis u tilizes the
in h eren t charge and m ass of m acrom olecules, enabling the sep aratio n and
id en tificatio n of many individual p ro te in com ponents in the fluid (O 'Farrell,
1975).
P ro sta tic fluid h as been analyzed previously by electro p h o retic m ethods.
E arlier investigations have id en tified im m unoglobulins, serum com plem ent
com ponents, tran sferrin , acid phosphatase and la c ta te dehydrogenase in the
fluid (G rayhack e t al., 1977a, 1977b, 1979). O th ers have had som e success
enriching the fluid for p ro sta te asso ciated com ponents by im m unoaffinity
chrom atography using antibodies to whole serum p ro tein s (D erm er, Silverm an
and Chapm an, 1982). Both C a rte r and R esnick (1982) and D erm er e t aL (1982)
have p erfo rm ed 2D gel analyses o f p ro sta tic fluid, and th e ir resu lts support the
findings discussed here. This c h ap ter re p o rts fu rth e r c h a ra c te riz a tio n of
p ro sta tic and serum p ro tein s in p ro sta tic fluid. Since th e p ro sta tic fluids used
in this study w ere co llected from re se c te d tissues, th ere was a strong
likelihood th a t residual blood w ithin the tissues was m ixed w ith the fluids.
P ro sta tic flhid p ro tein s sep arated on 2D gels w ere e lectro p h o retic ally tran s
ferred , or W estern b lo tte d , onto n itrocellulose sheets. The b lo ts w ere tre a te d
w ith antibodies recognizing various serum p ro tein s to lo cate and identify
serum antigens w ithin p ro sta tic fluid. The antigens w ere visualized by
39
------------------------------------------------------------------------- YZ5
autoradiography a fte r tre a tm e n t w ith ( I)-protein A, which binds th e
im m une com plexes form ed on th e b lo ts (Towbin e t al., 1979; B urnette, 1981).
This technique allow ed the id en tificatio n o f five serum p ro tein s in p ro sta tic
fluid, th ree of which w ere m ajor com ponents visualized by C oom assie Blue on
2D gels. T hree o th er p ro sta tic fluid p ro tein com ponents w ere d e te c te d th a t
had unique m olecular w eight and iso electric point ranges and did n o t share
antigenic site s w ith serum p ro tein s. These th re e non-serum com ponents
appear to b e p ro s ta te associated.
40
RESULTS
P ro sta tic fluids w ere co llected from m ore than tw enty hum an benign
p ro sta te hyperplasia (BPH) specim ens. The tissues w ere delivered from the
operating room to the lab o rato ry im m ediately a f te r p ro sta tec to m y . Speci
m ens w ere usually pink and w ere fairly firm , b u t flexible. O ccasionally a hard
w hite calciferous area, known to occur in BPH tissue (Prout, 1977), was
encountered. From a typical 6 cm p iece of tissue, approxim ately 100 to 200
p.1 of fluid was obtained by slicing th e tissue to a depth o f 3 to 4 mm and
co llectin g th e accum ulated liquid a t th e cu t su rface. Some specim ens
contained large q u a n titie s of fluid and yielded up to 500 Jll. R arely, tissues
w ere very dry and contained little or no fluid. P ro sta tic fluid appeared milky,
opaque, and slightly yellow in color, app aren tly due to the p resen ce of lipids
(Schaffner, 1981). A fte r low speed cen trifu g atio n , a sm all p e lle t containing
red blood cells was invariably obtained. The id en tity of the cells was
confirm ed by m icroscopic observation. The red blood cell p e lle t co n stitu ted
from 1 to 5% of the to ta l volum e of p ro sta tic fluid. Since plasm a contains
approxim ately 50% red blood cells by volum e, this den o tes a 2 to 10%
co n tam in atio n by blood. Fluids containing over 10% blood w ere discarded.
P ro sta tic fluids w ere analyzed by double im m unodiffusion against various
rab b it antibodies to hum an serum p ro tein s. When te sted against antibodies to
hum an a - l- a c id glycoprotein, a -1-an tich y mo trypsin, ot— 1 -p ro tein ase inhibitor
and haptoglobin, p ro sta tic fluid form ed strong p recip itin lines (Fig. 2-1). The
lines showed ap p aren t re a ctio n s of id en tity b etw een th e antigens p re sen t in
serum and p ro sta tic fluid. No p re c ip itin lines w ere form ed w ith a n ti-a -l-B
41
glycoprotein and an ti-a-2 -H S glycoprotein. T h erefo re, not all serum p ro tein s
are p resen t in su fficien t q u a n titie s to be d e te c te d by this m ethod.
P ro sta tic fluids w ere analyzed by tw o dim ensional gel electrophoresis
using various wide and narrow iso elec tric focusing pH ranges in the first
dim ension and 10% polyacrylam ide gels in th e second dim ension. A 10%
polyacrylam ide gel resolves com ponents w ith m olecular w eights ranging from
about 20,000 to 200,000 daltons. As discussed below , th e iso electric points
noted here are described as apparent due to the presence of 8 M u re a in the
first dim ension. Likew ise, m olecular w eights are designated as ap p aren t
because th e m obility of glycoproteins in electro p h o resis is not alw ays a
re fle c tio n of tru e m olecular m ass.
When analyzed on a 2D gel using th e broad pH range of 3.5 to 10 and
stained for p ro tein using Coom assie Blue dye, p ro sta tic fluid resolved into
sev eral p ro tein groups as seen in Fig. 2-2. When fluid was run on a narrow er
pH range, pH 5 to 7, th e p a tte rn s of th e m ajor p ro tein s seen in th e n e u tra l and
acidic portions of the gel in Fig. 2-2 w ere expanded and resolution was
im proved (Fig. 2-3). These m ajor com ponents are designated as I through VII.
Fig. 2-4 illu stra te s how m inor com ponents b ecam e visible when g re a ter
am ounts of p ro sta tic fluid w ere loaded on a 2D gel. This investig atio n focuses
on the m ajor com ponents seen in Fig. 2-3 th a t have e stim ate d co n cen tratio n s
of 0.2 m g/m l, or g re a te r. Many of th ese p ro tein s m igrated as groups,
producing sets of spots positioned side by side. The m ore acidic spots, or
isotypes, had slightly higher m olecular w eights. This p a tte rn is o ften
c h a ra c te ristic of glycoproteins. When a se p a ra te gel was stained for carbohy
d rates using th e periodic acid-S chiff's reag en t stain (PAS reactio n ), com po
42
nents I through V stained pink, indicating th a t th ey are glycoproteins (Fig. 2-
5).
The resolution of p ro sta tic fluid com ponents obtained on 2D gels of pH 4
to 6 range was very sim ilar to th a t obtained w ith pH 5 to 7 range. The p ro tein
groups seen in Fig. 2-3 sh ift to the rig h t, w ith th e exception of com ponent I
which rem ains as an unresolved sm ear on th e acidic side of th e gel.
When norm al hum an serum was rim on a pH 5 to 7 range 2D gel and
stained w ith C oom assie Blue (Fig. 2-6), th e c h a ra c te ristic p a tte rn of serum
p ro tein s and glycoproteins was seen as previously id en tified by A nderson and
Anderson (1977). Serum glycoproteins resolved on 2D gels also stain ed pink by
the PAS reactio n .
When the 2D gel p a tte rn s of p ro sta tic fluid (Fig. 2-3) and serum (Fig. 2-
6) w ere com pared, it was seen th a t p ro sta tic fluid com ponents had ranges of
m olecular w eights and iso elec tric points (pi's) which w ere sim ilar to th e values
of sev eral com ponents in serum . For exam ple, p ro sta tic fluid com ponents II,
IH and V m igrated to positions sim ilar to serum haptoglobin, and group IV
trav eled to a position n ear a -l-p ro te in a s e inhibitor. G roups VI and VH w ere
lo cated in th e area n ear serum album in. These observations, along w ith
im m unodiffusion resu lts described above, led to th e question of w hether any
p ro sta tic fluid com ponents visualized on 2D gels w ere serum p ro tein s. The
W estern b lo ttin g technique was used in a tte m p ts to answ er this question. A
series of W estern blots w ere p erfo rm ed on several 2D gels of p ro sta tic fluid
and serum , followed by im m unoautoradiography.
The lo catio n of a -l-p ro te in a s e inhibitor on 2D gels of serum was known
from previous work (Anderson and A nderson, 1977). The iso electric point
range and m olecular w eight w ere id en tified as p i 5.3-5.5 and 54,000 daltons on
43
th e gel in Fig. 2-6. Im m unoautoradiography p erfo rm ed on a W estern b lo t of a
serum -2D gel established th a t antibodies to a -l-p ro te in a s e inhibitor localized
this p ro tein a t th e ex p ected position on th e blot. The sam e antibodies
visualized a -l-p ro te in a s e inhibitor on a W estern b lo t o f a p ro sta tic fluid-2D
gel, and th e resulting autoradiograph is seen in Fig. 2-7. A dark spot was
found on the film corresponding to the pro p er m olecular w eight and
iso electric point range for a -l-p ro te in a s e inhibitor, 54,000 and p i 5.3-5.5,
respectively. a -l-P ro te in a s e inhibitor m ig rated to a position which has a
low er p i value and higher m olecular w eight than com ponent IV (Figs. 2-8 and
2-9). T herefore, p ro sta tic fluid com ponent IV is n o t a -l-p ro te in a s e inhibitor.
Albumin w as recognized by this antibody p rep aratio n on b lo ts of b o th serum
and p ro sta tic fluid, although the antibodies w ere claim ed by the m an u factu rer
to be specific for a -l-p ro te in a s e inhibitor. Also, these antibodies apparently
recognize two o th e r p ro sta tic fluid com ponents (Figs. 2-7 and 2-8). One has
m olecular w eight of 49,000 and p i o f 4.8 to 5.1, and the o th er has m olecular
w eight of 52,000 and p i o f 5.2 to 5.3. The 52,000 m olecular w eight com ponent
does not correspond to any of th e serum com ponents seen in Figure 2-6.
N either of th ese two com ponents w ere visualized on a W estern b lo t of serum .
The 49,000 m olecular w eight com ponent is slightly visible by Coom assie Blue
stain on a 2D gel o f p ro sta tic fluid. Both th e 49,000 and 52,000 m olecular
w eight com ponents are of higher m olecular w eights and low er p i’s than
p ro sta tic fluid com ponent IE , one of th e m ajor com ponents seen on 2D gels
(Fig. 2-9).
A ntibodies to haptoglobin w ere te ste d on W estern b lo ts of serum - and
p ro sta tic fluid-2D gels. Im m unoautoradiography o f th e serum b lo t visualized
both a and 3 subunit chains of haptoglobin (not shown). The autoradiograph of
44
th e p ro sta tic fluid blo t also showed th e presen ce of haptoglobin a and f 3
subunits. The a chains, seen at 20,000 m olecular w eight, did not correspond to
any p ro sta tic fluid p roteins seen on 2D gels stain ed w ith C oom assie Blue.
H ow ever, th e 0 chain visualized on th e p ro sta tic fluid W estern blot correspon
ded to p ro sta tic fluid com ponents HI and V in Fig. 2-9. Fig. 2-10 illu stra te s
how th e m igration p a tte rn s of IH and V m atch ed th e m igration of 3 chains on
th e im m unoautoradiograph of th e W estern b lo t. They are superim posable,
w hich in d icates th a t com ponents m and V are recognized by anti-haptoglobin
antibodies. Identical values for m olecular weigjhts, 41,000, and ranges of
iso elec tric points 5.4 to 5.9, as well as id e n tic al an tig en icities confirm s th a t
com ponents III and V are haptoglobin 0 chains. Also, album in was again
recognized on both serum and p ro sta tic fluid W estern blots.
A ntibodies recognizing a - 1-antichym otrypsin w ere used to lo c a te this
antigen on 2D gels of serum and p ro sta tic fluid, a-1-A ntichym otrypsin was
visualized on both serum - and p ro s ta tic fluid-W estern blots by im m unoauto
radiography. The resu lts w ere sim ilar to those obtained w ith a -l-p ro te in a se
inhibitor in th a t a - 1-ant ichym o try p sin does not correspond or co m ig rate w ith
any of th e com ponents of p ro sta tic fluid seen in Fig. 2-9. T h erefore, a-1-
antichym otrypsin is a m inor com ponent of p ro sta tic fluid or a contam inant
from m inor vascular disruption th a t occurs during fluid collection.
In control im m unoautoradiography ex p erim en ts, non-im m une rab b it
serum was su b stitu ted fo r im m une antibody prep aratio n s. None of th e proteins
in e ith er serum or p ro sta tic fluid w ere visualized on autoradiographs, in d icat
ing th a t non-specific binding of im m unoglobulins to proteins on th e blot does
not occur.
45
On all two dim ensional gels using pH ranges 3.5 to 10, 4 to 6 or 5 to 7, a -
1-acid glycoprotein (AGP) m ig rates as an unresolved sm ear of C oom assie Blue-
stained or PA S-stained m a te ria l on th e acidic side of th e gel. When serum is
run on a 2D gel using pH range 3.5 to 5 in the first dim ension, AGP resolves
into a group of nine d istin c t spots, or isotypes (see Fig. 5-1). M ost serum
p ro tein s are excluded from this pH range and do not e n te r the gel. Serum AGP
isotypes alw ays m aintain a c h a ra c te ristic , easily identified p a tte rn on 2D gels.
When AGP isotypes w ere W estern b lo tted , all isotypes w ere recognized by
antibodies to AGP (Fig. 2-11). All nine isotypes are visualized on the
autoradiograph, identifying them antigenically as AGP.
When p ro sta tic fluid was run on 2D gels of pH range 3.5 to 5, th e
c h a ra c te ristic isotype p a tte rn of serum AGP was seen by C oom assie Blue
staining (Fig. 2-12). O ccasionally, dilute perch lo ric acid tre a tm e n t of pros
ta tic fluid was used to p re c ip ita te substances th a t in te rfe re d w ith 2D gel
electrophoresis. This allow ed good reso lu tio n o f AGP iso types, since all
isotypes rem ained in solution a f te r the acid tre a tm e n t. By im m unoautoradio
graphy of W estern blots m ade from 2D gels of all pH ranges, antibodies to
AGP recognized this com ponent in p ro sta tic fluid.
In sum m ary, com m ercially available antibodies to serum p ro tein s reco g
nized the following com ponents on 2D gels of serum : album in, a -l-p ro te in a s e
inhibitor, a -1 -a n t ichym o trypsin, haptoglobin a - and B-chains, and a - l- a c id
glycoprotein. These antibodies also recognized the sam e serum com ponents on
2D gels of p ro sta tic fluid. H ow ever, they did n o t recognize th re e of th e m ajor
glycoproteins of p ro sta tic fluid th a t are seen on 2D gels, p ro sta tic fluid
com ponents II, IV and VII.
46
Fig. 2-1. Double im m unodiffusion of p ro sta tic fluid and serum against
antibodies to serum p ro tein s. C ontents o f th e w ells: w ell A, antibodies to a -1 -
acid glycoprotein; w ell B, antibodies to a -l-p ro te in a s e inhibitor; w ells C and
F, p ro sta tic fluid; w ell D, antibodies to a -1-a n t ichym o trypsin; w ell E, an ti
bodies to haptoglobin; w ell G, norm al serum .
Fig. 2-2. Two dim ensional gel electrophoresis of 30 (J.1 of p ro sta tic fluid. The
pH range for iso electric focusing w as 3.5-10. P ro tein s w ere visualized by
staining th e gel w ith Coom asie Blue.
47
Figure 2-1
IEF
molecular
weight
T
1
- 6 8 kd
- 4 3 kd
- 3 0 kd
% t
Figure 2-2
Fig. 2-3. Two dim ensional gel electro p h o resis o f 5 |il of p ro sta tic fluid. A pH
range o f 5 to 7 w as used in the iso electric focusing dim ension and p ro tein s
w ere stained w ith C oom assie Blue. The seven m ajor p ro tein groups are labeled
I through VII. AGP, a - l- a c id glycoprotein; ALB, album in.
Fig. 2-4. Two dim ensional gel electro p h o resis of 20 |ll o f p ro sta tic fluid using
a pH range 5 to 7 for iso electric focusing. P ro tein s w ere visualized w ith
C oom assie Blue. The labeling schem e is th e sam e as in Fig. 2-3.
49
I D
0
<
CL
1
C O
o
C O
ACIDIC
I
AGP
I
ISOELECTRIC FOCUSING
ffT
ALB
I
n
in
Figure 2-3
Iff
fftt
&
*
MOLECULAR
WEIGHT
- 94,000
- 68,000
4 $ - 4 3 ,0 0 0
* - 30,000
m a l K I -21.000
DYE FRONT
C D
<
a
i
co
a
co
i
II
IEF
III V
VI
-VII
IV
molecular
weight
-68 kd
-43 kd
-30 kd
Fig. 2-5. Two dim ensional gel electro p h o resis of 5 Jil o f p ro sta tic fluid, using
a pH range 5 to 7 for iso elec tric focusing. G lycoproteins w ere stain ed by the
periodic acid-S chiff's reactio n .
Fig. 2-6. Two dim ensional gel electro p h o resis of 10 p,l o f norm al serum , using
a pH range 5 to 7 for iso elec tric focusing. P ro tein s w ere stained w ith
C oom assie Blue. AGP, a - l- a c id glycoprotein; Achy, a -l-a n tic h y m o try p sin ; PI,
a -l-p ro te in a s e inhibitor; Hp, haptoglobin $ -chains; ALB, album in.
51
CD
5
I
CO
a
co
Figure 2 - 5
acidic
Aci h y p i
agp f y
I T
1
IEF
IV
IEF
molecular
w eight
I - 6 8 kd
- 4 3 kd
- 3 0 kd
basic
^ -
♦ * ♦ • ♦ » • ]
ALB
molecular
weight
- 9 4 kd
- 6 8 kd
k * - 4 3 kd
Hp
I
- 3 0 kd
Figure 2-6
52
Fig. 2-7. The lo cation of a -l-p ro te in a s e inhibitor on two dim ensional e le c tro
p h o retic gels as d eterm ined by im m unoautoradiography of a W estern b lo t. The
blot was obtained from a 2D gel of p ro sta tic fluid. A pH range of 4 to 6 was
used for iso elec tric focusing. The blot was tre a te d w ith antibodies to a -1 -
125
p ro tein ase inhibitor and ( 3)-protein A. PI, a -l-p ro te in a s e inhibitor; ALB,
album in.
Fig. 2-8. C om posite m ap localizing a -l-p ro te in a s e inhibitor by superim posing
th e W estern blot autoradiograph in Fig. 2-7 over th e C oom assie B lue-stained
p ro te in p a tte rn in Fig. 2-9. Open circles rep resen t C oom assie B lue-stained
p ro tein s th a t did not coincide w ith darkened areas on th e W estern b lot. The
filled circles re p re se n t C oom assie B lue-stained p ro tein s th a t coincided w ith
darkened spots on th e W estern b lot. S triped areas rep resen t darkened regions
of the autoradiograph th a t did not overlap w ith C oom assie B lue-stained
p ro tein s. PI, a -l-p ro te in a s e inhibitor.
53
IEF
cs
<
Q.
I
CO
a
CO
Figure 2-7
y
molecular
weight
-68 kd
-43 kd
-30 kd
IEF
ALB
o Q o
,fOQOOOO o o
° OOOo o o
CO
Figure 2 - 8
54
Fig. 2-9. Two dim ensional gel electro p h o resis o f p ro sta tic fluid using a pH
range o f 4 to 6 for iso electric focusing. P ro tein s w ere stained w ith Coom assie
Blue. AGP, a - l- a c id glycoprotein; ALB, album in.
Fig. 2-10. C om posite map localizing haptoglobin $-chain on 2D gels by
superim posing a p ro sta tic fluid W estern b lo t autoradiograph w ith a p ro sta tic
fluid p ro te in p a tte rn . The pH range for iso electric focusing w as 4 to 6 . The
125
W estern b lo t was tre a te d w ith antibodies to haptoglobin and ( I)^protein A.
P ro tein s w ere visualized w ith C oom assie Blue. Open circles, filled c ircles and
strip ed a re as are defined in th e legend to Fig. 2-8.
55
SDS-PAGE
IEF
AGP
I
II
III
Figure 2 - 9
molecular
weight
- 6 8 kd
- 4 3 kd
-3 0 kd
IEF
CD
<
Q-
I
CO
CO
VI
ALB
A
0
0 ° ° o = | V
i
_--r- O O Q Q © o O
II 0 ( 9 $ £ E > $ T f f > — y
III
Figure 2 -1 0
56
Fig. 2-11. The lo calizatio n of a - l- a c id glycoprotein on a W estern b lo t of
norm al serum by im m unoautoradiography. A pH range o f 3.5 to 5 w as used for
iso elec tric focusing. The b lo t w as tre a te d w ith antibodies to a - l- a c id
125
glycoprotein and ( I)-protein A. The AGP isotypes are labeled 1 through 9
(also see Fig. 5-1).
Fig. 2-12. Two dim ensional g el electro p h o resis of p ro sta tic fluid, using a pH
range o f 3.5 to 5 for iso elec tric focusing. P roteins w ere stained w ith
C oom assie blue. Only isotypes 2 through 8 are visible. Isotype 1 and 9 are
below the level o f d e tec tio n o f C oom assie Blue stain on this g el (see Ch. V).
57
SDS-PAGE SDS-PAGE
IEF
molecular
w e ig h t
Figure 2-11
1 2 3 4 5 6
( t t * * 7 8 g
l r r
- 6 8 kd
- 4 3 kd
- 3 0 kd
IEF
J 4 / i t II
2 J 4 5 6 7 8
Figure 2 -1 2
molecular
weight
- 6 8 kd
- 4 3 kd
-3 0 kd
58
DISCUSSION
The purpose of this study w as to in v estig ate the range of m acrom ole
cules p re sen t in p ro sta tic fluid by use of th e high resolution technique, tw o
dim ensional gel electrophoresis. This m ethod resolved p ro sta tic fluid p ro te in s
into seven m ajor com ponents, visible by C oom assie Blue staining on 2D gels.
The iso elec tric points of resolved p ro te in s and glycoproteins are described as
apparent due to the p resen ce of 8 M u re a in first dim ension gels. This
ch aotropic ag en t a lte rs th e p i values as com pared to o th er m ethods of
determ ining iso electric p o in ts (R ig h etti and C aravaggio, 1975; O’F arrell,
1975). Sim ilarly, m olecular w eights are described as ap p aren t to em phasize
th a t m olecular w eight d eterm in atio n s in S D S -electrophoresis is a com bination
of m obility in th e e le c tric field and the filterin g e ffe c t of th e polyacrylam ide
m atrix. M obility is not n ecessarily a d ire c t m easure o f tru e m olecular w eight.
This is p a rtic u la rly evident w ith glycoproteins. T heir m obility can b e a lte re d
by th e p resen ce o f carb o h y d rates which m ay change the size and shape o f the
SD S-glycoprotein com plex and a lte r th e num ber of SDS m olecules bound p e r
glycoprotein m olecule (Frank and Rodbard, 1975).
Some glycoproteins sep a ra te on 2D gels as a group of closely m igrating
spots, which display a gradual in crease in m olecular w eight w ith decreasing p i
values. This m igration p a tte rn is due to carbohydrate h etero g en eity in which
v ariab le am ounts o f n eg ativ ely charged sialic acids and n eu tral sugars are
a tta c h e d to th e polypeptide backbone (A nderson and Anderson, 1977; 1979).
C harge v ariatio n s resu lt in iso elec tric point h etero g en eity w here th e m ost
acidic spots have th e g re a te s t am ounts of sialic acids a ttach ed . F or exam ple,
when serum haptoglobin 3 -chains are tre a te d w ith neuram inidase to rem ove
59
sialic acids, th e num erous spots all sh ift to a single, m ore basic iso electric
p o in t (Anderson and Anderson, 1979)> re fle c tin g the singular p i of the
polypeptide backbone. S everal of th e p ro sta tic fluid com ponents, such as
com ponent II and com ponent IV show such p i and m olecular w eight h e te ro
geneity. T h at com ponents I through V are glycoproteins is also confirm ed by
th eir ability to stain by th e PAS reactio n .
Most previous re p o rts studied p ro sta tic fluid expressed from th e gland by
dig ital m assage and co llected from the u re th ra l m eatus. This m ethod has the
disadvantage of m ixing p ro s ta tic fluid w ith urine, sperm atozoa and secretio n s
of o th er accessory sex organs such as sem inal fluid (G rayhack and Lee, 1981).
O thers have trie d to d escribe p ro sta tic com ponents in sem inal fluid or in split
e jac u la te s (Zaneveld and Tauber, 1981), b u t this is unsuitable since p ro sta tic
fluid only com prises about 30% to 40% of e jacu late volum e (Eliasson, 1976;
K halil e t al., 1979). The m ethod o f co llectin g p ro sta tic fluid d irectly from
re se c te d p ro sta te overcom es th e problem s of m ixing p ro sta tic fluid w ith the
p ro d u cts o f gonads or o th e r accessory sex organs. How ever, th e re was
le g itim a te concern th a t th e m ethod o f co llectin g p ro sta tic fluid by th e slicing
o f re se c te d hyperplastic p ro s ta te tissu e resu lted in con tam in atio n by residual
blood w ithin th e organ. The m ixing of blood w ith p ro sta tic fluid was
confirm ed by tw o observations. F irst, red blood cells w ere p elle ted from
freshly co llected fluid. Second, double im m unodiffusion d e te c te d a - l- a c id
glycoprotein, a -l-p ro te in a s e inhibitor, a -l-a n tic h y m o try p sin and haptoglobin
in p ro sta tic fluid.
T h erefore, th ere w as a need to su b tra c t th e background of serum
p ro tein s from th e 2D gel p a tte rn to establish which com ponents are unique to
th e p ro sta tic fluid. A nderson and A nderson (1977) have done extensive
60
c h a ra c te riz a tio n o f serum and plasm a p ro tein s via tw o dim ensional gel
electro p h o resis. Subsequent work disclosed th a t e lectro p h o retically sep arated
p ro te in s re ta in th e ir an tig en icity and can b e im m unologically d e te c te d e ith e r
in polyacrylam ide gels (G ranger and L azar ides, 1979) or on W estern b lo ts
(Towbin et al., 1979; B urnette, 1981). T h erefo re, 2D gel electro p h o resis,
W estern b lo ttin g and im m unological d e tec tio n w ere com bined in this study to
id en tify serum p ro tein s p re sen t in p ro sta tic fluid. The th re e independent
p a ra m e te rs of iso elec tric point, m olecular w eight and antig en icity serve to
id en tify com ponents sep arated by this m ethod (A nderson and Anderson, 1977).
The in itia l a tte m p ts to lo cate antigens sep arated on 2D gels w ere
p erfo rm ed on th e gel itse lf w ithout W estern b lo ttin g , following the technique
d escribed by G ranger and L azarides (1979). The polyacrylam ide gel containing
th e resolved antigen w as soaked in a d ilu te antibody solution. A fte r washing
125
aw ay excess im m unoglobulin, ( I)-protein A was used to localize im m une
com plexes via autoradiography. A m ajor problem w as th e very lengthy
125
incubations needed to allow antibodies and ( I)-protein A to infuse into the
1.5 m m -thick gel m atrix . High backgrounds o fte n resu lted on autoradiographs.
W estern b lo ttin g o f p ro te in and glycoprotein antigens from po ly acry l
am ide gels onto very thin nitro cellu lo se sh eets re ta in s the fidelity of 2D gel
sep aratio n and g re a tly im proves accessib ility o f antigens, allow ing m ore rapid
incubations and decreased non-specific background on autoradiographs. E lec
tro p h o retic tra n sfe r o f p ro te in s sep arated by 2D g el electro p h o resis was m ore
rapid for com ponents o f low er m olecular w eight and low er pL Slower tra n sfe r
o f high m olecular w eight com ponents w as noted previously (B urnette, 1981)
and probably re fle c ts th e re la tiv e ease w ith which sm all p ro tein s can m ig rate
through th e polyacrylam ide m atrix . Since tra n sfe r occurs a t pH 8.3, the m ost
61
acidic com ponents will h av e a g re a te r num ber o f n eg ativ e ch arg es p e r
m olecule which likely allow s m ore rapid m ig ratio n tow ards th e anode. How
ev er, electrophoresis for one hour a t 2 0 0 m illiam ps w as su ffic ie n t to tra n sfe r
all serum and p ro s ta tic fluid p ro tein s onto blots, as evidenced by subsequent
lack of C oom assie B lue-stained p ro te in in th e polyacrylam ide gels. A ntigens
bound to the n itro cellu lo se sh eet w ere easily d e te c te d by autoradiography
using th e app ro p riate rab b it an tibodies and radioiodinated p ro te in A, which has
high affin ity for th e F c p o rtio n o f rab b it im m unoglobulins (K ronvall e t al.,
1970).
C om m ercially available rab b it antibody p rep aratio n s allow ed id e n tific a
tion of five serum p ro te in s on W estern b lo ts o f norm al serum . All of these
com ponents w ere easily id en tified by an tig en icity as w ell as by 2D gel
m ig ratio n p a tte rn s id en tical to those determ in ed by A nderson and A nderson
(1977). A co n tro l W estern b lo t using non-im m une rab b it serum showed no non-
125
specific binding of rab b it Ig or ( I)-p ro tein A to any serum com ponents on
th e blot.
When th ese antibody p re p a ratio n s w ere te ste d on W estern b lo ts of
p ro s ta tic fluid-2D gels, the sam e five serum p ro tein s w ere seen. Of these
five, th ree are iden tified as m ajor p ro te in com ponents th a t are seen on 2D
gels. By m olecular w eight, iso elec tric point and an tig en icity , p ro s ta tic fluid
com ponents H E and V w ere id en tified as haptoglobin $ chain, com ponent I as 01-
1-acid glycoprotein, and com ponent VI as album in. The o th e r tw o serum
p ro te in s found, a -l-a n tic h y m o try p sin and a - 1 -p ro tein ase inhibitor, w ere n o t
m ajor com ponents o f p ro s ta tic fluid. C om ponents II, IV and VH o f p ro sta tic
fluid w ere n o t recognized by any of th e antibodies used h ere. T h erefo re, since
th ese com ponents have d iffe re n t pi's, m olecular w eights, and an tig en icity th an
62
known serum p ro tein s, the conclusion is th a t they are u n re la ted to serum
com ponents.
a -1 -P ro te in ase inhibitor and album in on p ro sta tic fluid-2D gels have
higher ap p aren t positions as com pared to th e ir positions on serum -2D gels.
This is a p ro tein loading e ffe c t due to sm aller am ounts of th ese p ro tein s
p re sen t in p ro s ta tic fluid th an p re se n t in serum . As a g re a te r am ount of
p ro tein is localized in one spot th e re is, in general, an assy m etric expansion of
the spot tow ards th e b o tto m o f the g el (O 'F arrell, 1975).
An in te re stin g re su lt w as found on th e p ro sta tic fluid-W estem b lo t which
was tre a te d w ith antibodies to a -1 -p ro tein ase inhibitor. This antibody
p re p a ratio n recognized tw o com ponents in th e p ro sta tic fluid which w ere not
recognized on a serum b lo t. The com ponent o f 52,000 m olecular w eight (PF52)
had a unique m olecular w eight and pi, and did n o t resem b le any serum p ro tein
d e te c te d by these m ethods. The com ponent o f 49,000 m olecular w eight (PF49)
appeared to m ig rate to th e p o sition of serum a-2 -H S glycoprotein. H ow ever,
this com ponent was not recognized by th ese antibodies on a W estern b lo t of
serum , indicating th a t PF49 is n o t a-2 -H S glycoprotein. N evertheless, PF52
and PF49 w ere bound by antibodies recognizing a - 1 -p ro tein ase inhibitor. PF49
and PF52 do n o t share m o lecular w eight or p i c h a ra c te ristic s w ith ot— 1 —
p ro tein ase inhibitor. These tw o p ro sta tic fluid com ponents could b e cross
re a c tiv e w ith th e inhibitor. A ltern ately , they m ay be m odified form s or
breakdow n p ro d u cts o f a - 1 -p ro tein ase inhibitor containing a lte re d carbohy
d ra te chains or p ro te o ly tic ally processed polypeptide backbones. Such m odifi
cations o f glycoproteins can be resolved by tw o dim ensional gel electro p h o
resis as changed m olecular w eight or changed p i or both. A dditionally, the
antibodies to a - 1 -p ro tein ase inhibitor recognized o th e r p ro tein s on th e b lo t of
63
n orm al serum , for exam ple, album in. It is possible th a t PF49 and PF52 are
breakdow n pro d u cts o f one o f th ese com ponents. In the absence o f a m ore
specific antibody p rep aratio n , any relationship of PF49 and PF52 to p ro te a se
inhibitor cannot be clearly established.
None o f th e com m ercially available rab b it antibody p re p a ratio n s was
singularly specific for one antigen on W estern b lo t im m unoautoradiography.
Only in th e less sensitive double im m unodiffusion assay did th ese antibodies
appear specific for a single antigen. Im m unoautoradiography was su fficien tly
sen sitiv e to allow reco g n itio n o f o th e r serum p ro tein s. A co n tro l ex p erim en t
using non-im m une rab b it serum did n o t visualize any hum an serum p ro tein s on
a W estern blot, indicating the lack of non-specific binding of ra b b it im m uno
globulin to b lo tte d p ro tein s. It is likely th a t o th er serum p ro tein s w ere
im p u rities in th e antigen p re p a ratio n s used to im m unize rab b its. Album in w as
visualized by all the a n tisera used. Its high levels in serum , over 50% o f to ta l
p ro te in (Putnum , 1975), m akes its rem oval p a rtic u la rly d ifficu lt during p u rifi
catio n of o th er serum com ponents. The antibody p rep aratio n s used h ere
d e te c te d from one to th re e e x tra serum com ponents besides th e one th ey w ere
alleged to bind specifically. The lack of sp ecificity was useful, how ever,
allow ing recognition o f album in on W estern blots. A disadvantage in th e case
o f a - 1 -p ro tein ase inhibitor is th a t it is im possible to d iscern w hat serum
p ro te in or p ro tein s are im m unologic ally cross re a c tiv e w ith PF49 and PF52.
Serum a - l- a c id g lycoprotein iso types did n o t clearly resolve on 2D gels
which used iso electric focusing range above pH 3.5 to 5, although iso types
began to sep a ra te on som e pH 4 to 6 range 2D gels. The lack o f resolution on
high pH ran g e 2D gels is due to tw o facto rs. F irst, AGP is o u t of its o p tim al
pH range for p ro p er isotype reso lu tio n (pH 3.5 to 5) and second, AGP m ig rates
64
very close to a sm ear o f SDS m olecules on th e second dim ension gel. The
source o f these m olecules is the first dim ension in which th e highly anionic
SDS, used in th e denaturing solution, focuses a t th e acidic end of the gel.
Subsequently, the SDS m ig rates as a stre ak in th e second dim ension. On 2D
gels using pH 4 to 6 and pH 5 to 7 ranges, AGP focuses n e a r th e SDS m olecules
which in te rfe re w ith its resolution and m igration in the second dim ension. The
low pH ran g e of 3.5 to 5 solves this problem by allowing AGP to m ig rate to
the c e n te r of the gel, aw ay from the SDS stre ak . This p e rm its ex cellen t
resolution of AGP into nine iso ty p es ranging from pH 3.9 to 4.4 and m olecular
w eights o f 39,000 to 48,000. A ntibodies to AGP reco g n ize a ll nine isotypes on
2D gels, which confirm s th e id en tity of th ese spots. P ro sta tic fluid AGP
appears id en tical to serum AGP, displaying the sam e isotypes.
W hat m ay b e th e significance of serum com ponents in p ro sta tic fluid?
G rayhack e t al. (1979) found IigG, IigA, IgM, tra n sfe rrin and com plem ent
com ponents in p ro s ta tic fluid th a t w as co llected through th e u re th ra a fte r
d ig ita l m assage. It is possible th a t serum p ro tein s n o n -selectiv ely diffuse into
th e p ro sta tic glandular lum ina and are subsequently found in p ro s ta tic fluid.
A ltern ately , selectiv e tran sp o rt may be occuring. N aritoku (1982) has
im m unohistochem ically localized im m unoglobulins in p ro sta tic epithelium and
concluded th a t these m olecules w ere synthesized by plasm a cells p rio r to th eir
p lacem en t into e p ith e lial cells. T herefore, a m echanism m ay occur in th e
p ro sta te sim ilar to th a t which occurs in b re a st w here the sec re to ry im m uno
globulin IgA is tran sp o rted from serum to m am m ary epithelium and u ltim a te ly
into m ilk (Lascelles, 1977). H ow ever, in the work discussed h ere, the
collectio n of p ro sta tic fluid from surgically re sec te d p ro sta te tissue re su lts in
co n tam ination of the fluid by blood. T herefore, the d a ta re p o rted in this
65
c h ap te r can n e ith e r support nor re fu te sele c tiv e tran sp o rt of serum p ro te in s
into p ro s ta tic fluid.
O f th e seven m ajor p ro te in com ponents resolved from p ro sta tic fluid on
2D gels, four, com ponents I, m , V and VI, have b een iden tified as serum
p ro te in s on th e basis of th e th re e p a ra m e te rs of ap p aren t pi, ap p aren t
m olecular w eight and an tig en icity . The o th e r th re e m ajor com ponents, II, IV
and VII, do not have im m unological cross re a c tiv ity w ith any serum p ro te in s
m igrating to sim ilar positions on 2D gels. O th er serum p ro tein s have b een
id en tified in sm all am ounts and none co -m ig rate w ith p ro sta tic fluid com po
nen ts II, IV, or VII. Two m inor com ponents o f p ro s ta tic fluid are id en tified as
im m unologic ally cross re a c tiv e w ith antibodies recognizing a - 1 -p ro tein ase
inhibitor and o th er serum p ro tein s. C onsidering the source of the p ro s ta tic
fluid and id e n tific a tio n of serum con tam in an ts, as w ell as th e apocrine and
m erocrine n a tu re of secretio n s from p ro s ta tic epithelium (Aum uller and A dler,
1979), it ap pears th a t th e th re e m ajor non-serum p ro te in s in p ro sta tic fluid are
p ro sta te -a sso c ia te d and are likely to b e ep ith elial cell products.
66
CHAPTER IE
CREATINE KINASE-BB IN PROSTATIC FLUID. PARTIAL PURIFICATION
AND IDENTIFICATION ON TWO DIMENSIONAL GELS OF PROSTATIC
FLUID.
67
INTRODUCTION
C reatin e kinase (EC 2.7.3.2, CK) c ata ly z es th e tra n sfe r of a high energy
phosphate from c re a tin e phosphate to adenosine diphosphate yielding adeno
sine trip h o sp h ate and c re atin e . It consists of tw o subunits of approxim ately
40,000 m olecular w eight th a t can com bine to form th re e activ e hybrids, MM,
MB and BB. These isozym es are distinguished by d iffe re n t m igration ra te s
when electro p h o resed on a cellulose a c e ta te m atrix . V arious isozym es
predom inate in diverse organ sites. Isozym e MM is p re v a len t in th e sk eletal
m uscle, MB in th e h e a rt and BB in th e brain. C re a tin e kinase - BB has also
been found in o th e r tissues: kidney, stom ach, thyroid, bladder, in te stin e,
u teru s, lung and p ro s ta te (Smith, 1972; Tsung, 1976). In p ro sta te it was
d e te c te d im m unohistochem ically in ep ith elial cells (Silverm an e t al., 1979).
F eld and W itte (1977) observed CK-BB en zy m atic a c tiv ity in sera of som e
p a tie n ts w ith p ro sta tic carcinom a, w hile such a c tiv ity w as u n d e tec ta b le in
sera of norm al individuals. H ow ever, using CK-BB iso lated from brain, a m ore
sensitive radioim m unoassay was developed w hich discovered m inute am ounts
of the enzym e in norm al hum an serum (Zweig e t al., 1978). U sing th is assay,
Silverm an e t al. (1979) d e te c te d e lev ated CK-BB in th e se ra of 15 of 17
u n tre a te d p ro sta tic carcin o m a p a tie n ts. They concluded th a t CK-BB is
released from e p ith e lial cells in benign and m alignant conditions and m ay
c o n trib u te to elev a te d enzym e lev els in the sera of carcin o m a p a tie n ts.
CK-BB has p a rtic u la rly high a c tiv ity in p ro s ta tic fluid o b tained from
h y p erp lastic tissue. It was found at a co n ce n tra tio n of 25,000 m U /m l, about
10,000-fold higher th an th e levels found in serum (Silverm an e t al., 1979).
This ch ap ter describes a schem e fo r p a rtia l p u rifica tio n of enzy m atically
68
activ e CK-BB from p ro sta tic fluid. In addition, th e m o lecular w eight and
iso e le c tric point range of the CK-B subunit is rep o rted . It is id en tified as a
re la tiv e ly m inor com ponent on tw o dim ensional gels of p ro sta tic fluid.
69
RESULTS
P ro sta tic fluid was te s te d fo r the presence of c re a tin e kinase-BB by
double im m unodiffusion ag ainst rab b it anti-C K -B B im m unoglobulins. A single
p re c ip itin line was form ed, confirm ing th a t the fluid con tain ed CK-BB antigen.
P ro sta tic fluid was su b jected to high speed c en trifu g atio n to rem ove
sm all p a rtic u la te debris. The resu ltin g su p ern ate was run on a Sephadex G-100
m olecular filtra tio n colum n, w hich yielded th e elution profile in F ig. 3-1.
P ro tein s, as d e te c te d by absorption at 280 nm , w ere sep a ra te d in to five m ajor
peaks. CK-BB en zy m atic a c tiv ity elu ted as a single p eak b etw een th e tw o
m ajor p ro tein peaks, ju s t behind th e colum n void volum e a t a point consistent
w ith th e CK m o lecu lar w eight of 80,000 daltons. The enzym e w as m onitored
by m easuring whole c re a tin e kinase activ ity . The a c tiv ity w as id e n tifie d as
predom inantly BB isozym e by cellulose a c e ta te electro p h o resis of a fra c tio n
o b tained from th e Sephadex G-100 colum n. This showed th a t over 95% of the
a c tiv ity m ig rated as BB isozym e (Fig. 3-2). F rac tio n s obtained from th e
Sephadex colum n, co ntaining enzym e a ctiv ity , w ere pooled and applied to a
D EA E-cellulose colum n. Bound anions w ere elu ted w ith a g rad ien t of 0 to 0.5
M N aCl in 0.1 M T ris-H C l b u ffer, pH 8.0, containing 0.1% $ -m ercap to eth an o l
(Tris-$M E). The CK-BB activ e peak elu ted a t 0.16 N aC l, equivalent to 320
m illim hos, in Tris-|3ME b u ffer (Fig. 3-3). H ow ever, th e a ctiv e enzym e was not
clearly se p a ra te d from o th er p ro tein s elu ted from th e colum n. Of 62,500 mU
of enzym e a c tiv ity placed on th e Sephadex G-100 colum n, 53,500 m U, or 8 6 %,
w ere reco v ered . From th e D EA E -cellulose colum n, on w hich 46,700 mU w ere
placed, 27,400 mU w ere elu ted , rep resen tin g 59% reco v ery .
70
An aliquot of p a rtia lly p u rified CK-BB w as rad io io d in ated to allow
visualization of m inute am ounts of p ro tein by 2D gel electro p h o resis and
autoradiography. The denaturing b u ffer used to p rep are sam ples for 2D gel
electro p h o resis se p a ra te s th e BB enzym e into individual B subunits of about
40,000 m o lecular w eight. A utoradiography displayed tw o se p a ra te p ro tein
groups having app ro x im ately 40,000 m olecular w eight, but having d ifferen t
iso e le c tric points. One se t of com ponents had pi above 5 on 2D gels, w hile the
o th er had pi below 4. Fig. 3-4 illu stra te s th e reso lu tio n of th e acid ic
com ponents in th e CK-BB p rep aratio n . On th is 2D gel of pH range 3.5 to 5,
nine se p a ra te spots w ere seen on th e acidic portion of th e gel. These spots had
th e sam e pi ran g e, m o lecular w eight range, and isotype d istrib u tio n p a tte rn as
serum a - l- a c id glycoprotein (AGP) visualized on 2D gels stain ed w ith C oom as-
sie Blue (Fig. 5-1). As described in C h ap ter II, AGP is p resen t in p ro sta tic
fluid in su fficien t am ounts to be seen on 2D gels. The acidic com ponent
illu stra te d in Fig. 3-4 is th e re fo re id e n tifie d as AGP. The m ore basic
com ponent view ed on th is radiograph w as CK-B subunit w hich w as b e tte r
reso lv ed on a 2D gel of higher pH range.
The p u rified B subunit of c re a tin e kinase, iso lated from brain by a
m ethod sim ilar to th e one described above (W. Shell, personal com m unication),
resolved in to five spots, or isotypes, on a 2D gel of pH range 5 to 7. The
C oom assie B lu e-stain ed CK-B isotypes are illu stra te d in Fig. 3-5. C om m erci-
125
ally available ( I)-CK-BB, also iso lated from brain, w as run on a 2D gel of
th e sam e pH range. The radiograph, illu stra te d in F ig. 3-6, showed th e sam e
reso lu tio n of CK-B in to 5 se p a ra te isotypes. The CK-B isotypes had pi range
125
of 5.8 to 6.1 and m o lecu lar w eight of 41,000 on 2D gels. ( I)-CK-BB w as
com bined w ith p ro sta tic fluid and su b jected to 2D gel electro p h o resis. The gel
__________ 71
was stain ed fo r p ro te in and subsequently autoradiographed. W hen th e resul-
125
ta n t film and th e C oom assie B lue-stained gel w ere superposed, ( I)-CK-B
was seen to co m ig rate w ith a group of spots lo c ated b etw een p ro sta tic fluid
125
com ponents IV and V, n o ted in Fig. 3-7. C o-m igration of ( I)-CK-B w ith
th e se p ro tein spots id en tified them as the c re a tin e kinase d e te c te d previously
in p ro sta tic fluid by enzy m atic and im m unologic assays. In addition, th is
estab lish ed th a t n e ith e r com ponent TV nor V is CK-B.
N euram inidase tre a tm e n t, w hich rem oves te rm in a l sialic acid m o ieties
125
from glycoproteins, did n o t a lte r th e 2D gel m ig ratio n p a tte rn of ( I)-CK-B,
in d icatin g th e absence of sialic acid. E n zym atically a c tiv e CK-BB elu ted from
th e Sephadex G-100 colum n did not bind to a colum n of C onconavalin A -
conjugated Sepharose 4B, denoting absence of m annose. Also, CK-BB did n o t
bind to w heat germ agglutinin-conjugated Sepharose 4B, w hich in d ic a tes lack
of N -acety l glucosam ine (Table 3-1). T h erefo re, CK-B does not appear to be
g lycosylated. P relim in ary re p o rts from am ino acid analysis of p u rified brain
CK-BB show ed th e p resen ce of a t le a st one phosphorylated am ino acid (Tokes,
unpublished observation). D iffere n t num bers of phosphorylated residues on
individual m olecules m ay acco u n t fo r th e m ultiple isotypes of CK-B ev id en t on
2D gels.
72
Table 3-1
L ectin affin ity chrom atography of CK-BB
Colum n m atrix
A m ount of
CK-BB
applied
(mU/m l)
A m ount of
CK-BB
reco v ered
(mU/ml)
p e rc e n t of a c tiv ity
th a t did not
bind to colum n
Sepharose 4B (control) 540 540 1 0 0 %
Con A -Sepharose 4B 540 532
99%
WGA - Sepharose 4B 540 517 96%
E nzym atically activ e CK-BB was obtained as a single peak from a Sephadex G-
100 chrom atography colum n. Equal am ounts of enzym e w ere p laced on th e
le c tin -a ffin ity colum ns and co n tro l colum n listed below . Colum n volum es w ere
1 ml. Unbound m a te ria l w ashed from colum ns w ere te ste d for CK-BB a ctiv ity
as described in M aterials and M ethods. P ositive co ntrols for th e WGA and
Con A colum ns w ere, resp ectiv ely , glycophorin and gp70 isolated from hum an
m ilk -fat-flo b u le m em brane (Imam, L aurence and N eville, 1981).
73
Fig. 3-1. Sephadex G-100 colum n chrom atography of p ro sta tic fluid 49,000 g
3
su p ern atan t. Bed volum e was 175 cm . E quilibration and elution b u ffer was
0.1 T ris/H C l, pH 8.0, containing 0.1% |3-m ercaptoethanol. P ro tein was
d e te c te d in efflu en t by absorbance at 280 nm . Enzym e a c tiv ity was d e te c te d
as described in M aterials and M ethods.
Fig. 3-2. D en sito m etric scan of CK isoenzym es a fte r electro p h o resis on
agarose film . Top) M igration of hum an CK-MM, CK-MB, and CK-BB isoen
zym es. B ottom ) M igration of CK isoenzym e e lu ted from th e Sephadex G-100
colum n in F ig. 3-1. CK enzym atic a c tiv ity is d e te c te d as described in
M aterials and M ethods.
74
Sephadex G-100
Prostatic Fluid Supernatant
.70
E 60
c
o
CO
™ .50
(-
<
LU
O .40
<
C O
o c
O .30
c o
20
<
5000
4000
3000
J .20
2000
1000
120 140 160 180 200 60 100 40 80 20
VOLUME (ml)
Figure 3-1
MM
STANDARDS
MB
BB
lli
o
Z
L U
O
C t)
o
DC
Z 5
_ l
L L
ORIGIN
PROSTATIC
ORIGIN
ELECTROPHORESIS
Figure 3-2
75
Fig. 3-3. D EA E -cellulose chrom atography of pooled activ e CK fractio n s
3
o b tain ed from th e Sephadex colum n. Bed volum e was 75 cm . E quilibration
and elu tio n b u ffer was 0.1 M T ris/H C l containing 0.1% $ -m ercap to eth an o l.
The g rad ien t w as produced by com bining in a g rad ien t m aker equal volum es of
th e b u ffer containing 0.5 JM N aC l and th e b u ffer alone. A sm aller volum e of
th e b u ffer containing 1.0 M ! N aCl was applied to th e end of th e lin ear g rad ien t.
P ro te in was d e te c te d by absorbance a t 280 nm . G radient was m easured by
conductivity (millimhos).
Fig. 3-4. Two dim ensional gel electro p h o resis of a rad io io d in ated CK-BB
p rep aratio n containing a - l- a c id glycoprotein. The pH range for iso e le c tric
focusing was 3.5 to 5. C om ponents w ere visualized by autoradiography.
76
DEAE C e llu lo s e
0.300
£
c
O
a 0.200
H -
<
r 40
ID
o
z
<
CD
C C
0
m 0.100
<
1
-30
Gradient 80 0 --2 0
600-
400--10
( - * - * I \
T 2 0 0 -
■ > « x x 4, ix -x -X "
1 1 1 —
100 200 300
400 500 600 700 800 900 1000 1100
EFFLUENT VOLUME (m l)
Figure 3-3
IEF
molecular
weight
CD
<
Q.
I
C/9
a
c o
t t
7 8
- 68 kd
-4 3 kd
-30 kd
Figure 3-4
77
Fig. 3-5. Two dim ensional gel electro p h o resis of p u rified c re atin e kinase-BB
(20 yg). The pH range fo r iso e le c tric focusing w as 5 to 7. P ro te in was
visualized by C oom assie Blue.
Fig. 3-6. Two dim ensional gel electro p h o resis of radioiodinated CK-BB. The
pH range fo r iso e le c tric focusing w as 5 to 7. R adioiodinated p ro tein was
visualized by autoradiography.
78
IEF
molecular
weight
C D
2
I
C O
a
co
- 6 8 kd
-4 3 kd
-30 kd
Figure 3-5
IEF
molecular
weight
C D
<
Q.
I
C O
a
co
-6 8 kd
-4 3 kd
-30 kd
Figure 3-6
79
IC C
CD
<
Q _
I
CO
o
co
— 1 L 1
VI
> 1 -VII
1
11 . * - I V
* 2 'C K - B
___ _ A * Sta.
III V
molecular
w eigh t
- 6 8 kd
- 4 3 kd
- 3 0 kd
Figure 3 -7
Fig. 3-7. Two dim ensional gel electro p h o resis of 20 y l of p ro sta tic fluid, using
pH range 5 to 7 fo r iso e le c tric focusing. The num bering schem e follows th a t
described in Ch. II. P ro tein s w ere visualized by C oom assie Blue. CK-B,
subunit of c re a tin e kinase BB isozym e.
80
DISCUSSION
C re atin e kinase BB was found previously in norm al and neo p lastic hum an
p ro sta te tissue by assay of isozym e a c tiv ity and by sp ecific im m unohisto-
chem ical staining (Tsung, 1976; Silverm an e t al., 1979; A leyassine and
M ad saac, 1980; K im ler and Sandhu, 1980; P retlo w e t al., 1982). CK-BB was
also p resen t a t high co n ce n tra tio n in p ro sta tic fluid (Silverm an e t al., 1979)
w here it could be d e te c te d by double im m unodiffusion. This isozym e was
e lev ated in th e sera of som e p ro sta te can cer p a tie n ts as m easured by a
radioim m unoassay using rab b it antibodies raised ag ain st brain CK-BB (Silver
m an e t al., 1979; H om burger e t al., 1980). Serum CK-BB in th ese p atie n ts
was also elev ated when assayed for enzy m atic a c tiv ity (Feld and W itte, 1977;
Coolen e t al., 1979; K im ler and Sandhu, 1980). CK-BB elev atio n was m ore
pronounced in u n tre a te d p a tie n ts (Silverm an e t al., 1979) th an in those
undergoing tre a tm e n t (Zweig and Van S teirteg h em , 1981). E levation of serum
CK-BB was also m ore pronounced in stag e D carcin o m a th an in o th e r stag es
(Feld e t al., 1980) and appeared to c o rre la te b est w ith poorly d iffe re n tia te d
carcinom a (A leyassine and M ad sa ac , 1980). To d a te, te s ts for elev ated serum
levels of CK-BB have proved m ore useful in identifying u n tre a te d , advanced
disease than in d e tec tin g e arly stag es of p ro sta te carcinom a.
This study shows th a t p ro sta tic fluid, o b tained from surgically re se c te d
h yp erp lastic p ro sta te s, can be used as a source of CK-BB. P ro sta tic fluid
contains CK-BB a t a co n cen tratio n of 25,000 m U /m l and th e re fo re is an
ex cellen t source of th is enzym e. H ow ever, a m ajor co n tam in an t of CK-BB
p rep ared from p ro sta tic fluid was a - l- a c id glycoprotein, w hich is known to
ex ist in p ro sta tic fluid obtained from re se c te d tissue (C hapter II). AGP elu tes
81
from DEAE ion exchange m atrices a t approxim ately th e sam e b u ffer ionic
stre n g th as does CK-BB (C. Csipke and W. Shell, personal com m unications).
CK-BB was d e te c te d on 2D gels of p ro sta tic fluid and resolved into
sev eral isotypes having various iso e le c tric points. A nother p ro sta tic enzym e,
p ro s ta tic acid phosphatase, has also been resolved by iso e le c tric focusing in to
m ultiple isotypes. The isotype p a tte rn was found to change in d iffe re n t
p ro sta te disease conditions (Chu e t al., 1977). As y e t, it is unknown w hether
th e isotype p a tte rn of CK-B sim ilarly changes in various p ro sta te diseases.
M onitoring changes in CK-B visualized on 2D gels m ay gain diagnostic re le
vance if a co rrelatio n is found b etw een isotype m igration p a tte rn s and
p ro s ta te neoplasia.
The CK-BB isozym e appeared elev a te d in sera of only th e m ost advanced
p ro sta te cancer cases, and th e elevation seem ed to c o rre la te b est w ith less
d iffe re n tia te d carcinom as. E levations w ere ra re ly seen in sera of p a tie n ts
w ith benign hyperplasia. A possible explanation for th e se observations m ay lie
in th e th re e dim ensional stru c tu re of the tissue. In h ealth y p ro s ta te and
h y p erp lastic tissue, glandular stru c tu re rem ains in ta c t and products are
seq u estered w ithin lum ina and ducts. In poorly d iffe re n tia te d carcinom a and
m e ta s ta tic lesions, acin ar stru c tu re is d estroyed and cellular products such as
CK-BB can be lo st into surrounding stro m a. E arly stag es or w ell d ifferen ti
a ted form s of can cer m ay show in te rm e d ia te am ounts of acin ar disruption.
Thus, p a tie n ts w ith benign or w ell d iffe re n tia te d neoplasia should display
n early norm al serum CK-BB levels w hile those w ith advanced or poorly
d iffe re n tia te d carcinom a should show CK-BB elevations.
CK-BB from p ro sta tic fluid was recognized by antibodies raised ag ainst
CK-BB from brain, in dicating an tig en ic sim ilarity of the m olecule from these
82
tw o sources. A lthough CK-BB from p ro s ta te has not y e t been shown to be
an tig en ically unique, antiserum raised against b rain CK-BB re a c te d less
strongly tow ards CK-BB p u rified from p ro s ta te (L. Silverm an, personal
com m unication). It is possible th a t p ro sta tic CK-BB contains som e unique
an tig en ic d eterm in an ts, since an o th er ubiquitous cellu lar enzym e, acid phos
p h atase, was found to be im m unologically d istin ct in th e p ro sta te (Shulman e t
al., 1964). H ybridom a technology could be u tiliz e d in a search fo r unique
antigenic d eterm in an ts on p ro sta te-d eriv ed CK-BB. H ighly sp ecific antibodies
m ay in crease th e sen sitiv ity and sp ecificity of im m unoassays for p ro sta tic CK-
BB in serum and m ay o ffer b e tte r d e tec tio n of early stag es of p ro sta te
carcinom a.
83
CHAPTER IV
ANALYSIS OF THE GLYCOPROTEINS SYNTHESIZED BY BENIGN HYPER
PLASTIC PROSTATE ORGAN CULTURES AND BY PROSTATE CARCINOMA
CELL LINE PC -3.
84
INTRODUCTION
P ro sta te tissue has been previously shown to rem ain viable in in v itro
cu ltu res for periods from a few days up to sev eral w eeks (for review see
H eatfield et al., 1980). Tissues respond to androgens to a very lim ited e x te n t
in cu ltu re and are easily m ain tain ed in androgen-free m edium (Franks, 1980).
Short term cu ltu res of benign h y p erp lastic tissue and epitheloid, carcin o m a-
derived cell lines synthesize and re le ase glycoproteins into cu ltu re m edia.
M etabolic iso tope-labeling of benign p ro sta te hyperplasia (BPH) specim ens and
autoradiography of labeled tissue sections showed th a t the ep ith elial cells are
th e m ain source of th e se m acrom olecules (Tokes and D erm er, 1977).
The work re p o rte d in this ch ap ter c h a ra c te riz e s th e glycoproteins pro
duced by p ro sta te organ cu ltu res and by a p ro sta te carcin o m a-d eriv ed cell
line. M acrom olecules w ere m etab o lically rad io lab eled by cu ltu rin g fresh BPH
tissue in th e presen ce of ra d io activ e carb o hydrate precursors. Sim ilarly, th e
p ro s ta te cell line PC -3 in trin sically radiolabeled glycoproteins when cu ltured
in th e presen ce of ra d io activ e carb o h y d rate or am ino acid precursors. Two
dim ensional gel electro p h o resis and fluorography w ere used to analyze th e
glycoproteins released by cu ltu res in to th e m edia and to com pare th e products
of the tw o cu ltu re system s. A dditionally, c u ltu re products w ere com pared to
p ro sta tic fluid glycoproteins resolved on 2D gels. Since th e gel m igration
p a tte rn s in d icated sim ilarities b etw een th e se com ponents and serum proteins,
th e biosynthesized com ponents w ere also analyzed by im m unoprecipitation
w ith antibodies to hum an serum p ro tein s. It was found th a t five glycoproteins
produced by BPH organ cu ltu res and five glycoproteins from PC -3 cell cu ltu re
have essen tially id en tical m olecular w eight and iso e le c tric point ranges. Addi
85
tionally, th re e of th e se five glycoproteins have th e sam e m olecular w eights
and iso e le c tric points as predom inant glycoproteins found in p ro sta tic fluid.
None of th e com ponents are im m unoprecipitable w hen te ste d against an ti
bodies to sev eral hum an serum p ro tein s. The evidence suggests th a t th ese
th re e com ponents are p ro sta tic products which are p laced into p ro sta tic fluid
in vivo.
86
RESULTS
F resh surgical specim ens of benign p ro sta te h yperplasia tissue w ere
14
in cu b ated fo r 24 to 48 hours in the presence of ( C )-glucosam ine to in trin sic
ally label glycoproteins. The m edia, containing glycoproteins released from
th e cu ltu res, was subsequently cen trifu g ed and extensively dialyzed to rem ove
debris and u n in corporated ra d io activ e precursors. Volume was reduced by
14
e ith e r lyophilization or co n cen tratio n in a Pro-D i-C on apparatus. When ( C)-
labeled glycoproteins w ere analyzed by 2D gel electro p h o resis, sev eral rad io -
lab eled groups w ere visualized fluorographically (Fig. 4-1). A com ponent
having m olecular w eight g re a te r th an 2 0 0 , 0 0 0 is seen a t th e top, and a stre a k
of poorly resolved m a te ria l is observed on th e acidic side of th e gel. Also
d e te c te d are seven groups of d istin ctly resolved glycoproteins. A com posite
14
m ap of th e ( C )-glycoproteins (Fig. 4-2) was draw n from six such fluoro-
graphs. N ote th a t seven of th e nine groups of glycoproteins resolved well on
2 D gels of pH range 5 to 7. These seven com ponents ranged in m olecular
w eights from 25,000 to 70,000 daltons and in iso elec tric points from 5.0 to 7.0.
14
The p ro sta te cell line PC-3 grow n in th e presence of ( C )-glucosam ine
also released radiolabeled m acrom olecules which w ere analyzed by 2D gels and
fluorography, shown in Fig. 4-3. The iso e le c tric focusing pH range of 4 to 6
afforded th e b est 2D gel resolution of PC -3 cell products. Sim ilar to BPH
cu ltu res, PC - 3 cells produced a high m olecular w eight com ponent, seen at the
top of th e gel, and a stre a k on th e acidic portion of th e gel. Eight
glycoprotein groups w ere resolved in th e c en te r of the gel. N ote the tw o
sm earing a rtifa c ts shown by arrow s in Fig. 4-3. These a rtifa c ts w ere caused
by n o n -rad io activ e p ro tein s, visible only on the C oom assie B lue-stained gel,
87
w hich w ere derived from fe ta l bovine serum added to th e cu ltu re m edium .
These serum p ro tein s m ig rated very n ear tw o radiolabeled PC -3 glycoprotein
14
products, slig h tly encoaching on th e ir positions. The o th er ( C )-glycoproteins
w ere u n a ffe cted . A com posite m ap, draw n from four fluorographs, illu stra te s
th e 2D gel sep aratio n of the released PC-3 cell products (Fig. 4-4). Nine
glycoprotein groups m ig rated to positions in m o lecular w eight range of 25,000
to 55,000 daltons and pi range of 4.0 to 6.5.
When the iso e le c tric points and m olecular w eights of th ese bio sy n th etic
products from organ and cell cu ltu res w ere tab u la ted , it was found th a t five
d ifferen t PC -3 and BPH p ro ducts share th ese physical c h a ra c te ristic s, liste d in
Table 4-1. These five glycoproteins w ere com pared to th e p ro sta tic fluid
com ponents visualized on C oom assie B lue-stained 2D gels (Fig. 2-3). T hree of
th e five biosynthesized glycoproteins share m o lecu lar w eight and iso elec tric
point ranges w ith com ponents found in p ro sta tic fluid (Table 4-2).
It was also no ted th a t biosynthesized glycoproteins from both organ and
cell cu ltu res shared m olecular w eight and iso e le c tric point ranges w ith several
serum p ro tein s. F or exam ple, a - l- a c id glycoprotein had pi range of 3.9 to 4.4
and m olecular w eight range of 40,000 to 48,000 (C hapter V), sim ilar to PC-3
com ponent 5 (Fig. 4-4). Likew ise, haptoglobin $ -chain had pi range 5.3 to 5.9
and 40,000 m o lecular w eight, very sim ilar to BPH com ponent VI (Fig. 4-2).
A dditionally, it was observed by double im m unodiffusion th a t antibodies recog
nized serum glycoproteins in p ro sta tic fluid (C hapter II), including haptoglobin
and a - l- a c id glycoprotein. T h erefo re, im m unoprecipitation ex perim ents w ere
p erfo rm ed to in v e stig ate w hether p ro sta tic cells or tissue in v itro produce
serum or serum -cross re a c tiv e glycoproteins.
88
T able 4-3 describes th e resu lts of im m unoprecipitation ex p erim ents
14
p erfo rm ed on ( C )-glucosam ine-labeled glycoproteins released by BPH cul
tu res. Im m unoprecipitation was also p erfo rm ed on radiolabeled pro tein s
released from hom ogenized BPH tissue. A ntibodies to hum an serum a - l- a c id
glycoprotein and haptoglobin failed to im m u n o p recip itate sig nificant am ounts
14
of ( C )-glycoproteins from e ith e r spent m edium or from hom ogenized tissue.
In 3 of 4 ex p erim en ts, non-im m une serum p re c ip ita te d g re a te r am ounts of
radiolabeled com ponents th an did im m une antibodies, in dicating th e non
sp ecific n a tu re of th ese in te ra ctio n s. In th e fo u rth exp erim en t, a n ti-h a p to
globin antibodies p re c ip ita te d slightly m ore m a te ria l th an did non-im m une
serum , b u t the d ifferen ce was not significant.
T able 4-4 shows th e resu lts of sev eral im m unoprecipitation exp erim en ts
14
p erfo rm ed on the ( C )-glucosam ine-labeled glycoproteins released from PC-3
cells. In in stan ces w hen m ore radiolabeled com ponents w ere p re c ip ita te d by
anti-A G P antibodies th an by non-im m une serum , th e d ifferen ce was not
sta tistic a lly significant. The tw o control exp erim en ts in Table 4-4 illu stra te
th a t the im m u n o p recip itatio n p rocedure w orked properly w ith th e ap p ro p riate,
radioiodinated antigen.
From th ese im m unoprecipitations of bio sy n th etic products, p e lle ts con
taining rad io activ e m a te ria l w ere soluabilized and run on 2D gels w hich w ere
subsequently fluorographed. None of th e resu ltin g fluorographs visualized any
com ponents a t th e m o lecular w eights and iso elec tric points corresponding to
th e serum glycoproteins recognized by th e antibodies used fo r im m unopre
cip itatio n . Thus, using antibodies to serum p roteins, im m unoprecipitations
w ere unsuccessful in sp ecifically recognizing any radiolabeled products of P C -
3 or BPH cu ltu res.
89
In addition to im m unoprecipitations, an im m unoaffinity chrom atography
ex p erim en t was p erfo rm ed . There was p a rtic u la r in te re s t in clarifying the
relationship of th e acidic PC -3 com ponent 5 and serum a - l- a c id glycoprotein.
N ote th a t com ponent 5 (Fig. 4-4) had a m igration p a tte rn very sim ilar to the
AGP com ponent on th e pH 4 to 6 range 2D gel of serum (Fig. 4-5). Initially,
PC -3 com ponent 5 was also resolved on a 2D gel using a pH g rad ien t of 3.8 to
5. The resu ltin g fluorograph (Fig. 4-6) visualized com ponent 5 as a group of
spots which has m oved fu rth e r to the rig h t on th e gel. C om ponent 5 appeared
a t 40,000 to 43,000 m o lecular w eight range and had a pi range of 4.1 to 4.5.
This again was very sim ilar to serum a - l- a c id glycoprotein which m ig rated on
this type of 2D gel a t 40,000 to 48,000 m olecular w eight range and a pi range
of 3.9 to 4.4 (C hapter V). The com ponent lab eled gp 42 was a poorly resolved
glycoprotein m igrating a t th e ex trem e acidic end of th e gel. M ost of th e
o th er, m ore basic PC-3 glycoproteins did not en ter into the first dim ension gel
and th e re fo re w ere n o t p resen t on this 2D gel.
To im prove sp ecific a c tiv ity of radiolabeled glycoproteins and to en
hance th e fluorography of acidic P C - 3 glycoproteins, th ese cells w ere cu ltu red
35
in the presence of ( S)-m ethionine. In addition, a m odified pH g rad ien t in the
first dim ension was used w hich re su lted in th e extended pH range of 3.5 to 5
and allow ed b e tte r 2D gel reso lu tio n of gp 42. The resu ltin g fluorograph of
3 5
( S)-labeled com ponents showed th a t all th e acidic glycoproteins which
14 35
previously in co rp o rated ( C )-glucosam ine also in co rp o rated ( S)-m ethionine
(Fig. 4-7). C om ponent 5 was again visible on th e rig h t side of th e fluorograph.
A dditionally, the low er pH g rad ien t resolved gp 42 (Fig. 4-6) in to a t le a st 6
d istin ct isotypes.
90
------------------------------------------------------------------------- 35----------------------------------------
D ialyzed, spent m edium containing re le ase d ( S)-labeled PC -3 glycopro
tein s was applied to an im m unoaffinity colum n of anti-A G P im m unoglobulins
co v alen tly coupled to Sepharose 4B. In a co n tro l exp erim en t, AGP p u rified
from hum an serum was successfully bound to th e colum n, subsequently eluted,
and found to m ain tain th e original AGP isotype p a tte rn on 2D gels, as in Fig.
35
5-1. The spent medium containing ( S)-labeled PC-3 glycoproteins was
c irc u la te d through th e colum n to allow su fficien t tim e for A G P-like antigens
to bind to th e antibodies coupled to th e colum n m atrix . A fterw ard s, unbound
m a te ria l was w ashed from th e colum n and bound ra d io activ e m olecules w ere
e lu te d w ith 3 JM potassium th io cy an ate in saline. The elu ted m a te ria l was
c o n ce n tra te d to reduce volum e and was run on a 2D gel of pH range 3.5 to 5 to
fluorographically visualize acidic PC-3 glycoproteins. No rad io lab eled acidic
glycoproteins of any kind w ere visualized on th e fluorograph. The only
ra d io activ e m a te ria l seen was an unresolved stre a k o rig inating a t the basic end
of th e first dim ension gel. A se p a ra te 2D gel of serum , run sim ultaneously,
visualized serum a - l- a c id glycoprotein, in d icatin g th a t ex p erim en tal condi
tions w ere su ffic ie n t to allow reso lu tio n of any acidic glycoproteins.
The 2D gel m ig ratio n p a tte rn s, illu stra tin g m olecular w eight and iso elec
tric point ranges of a - l- a c id glycoprotein and acidic PC -3 glycoproteins, are
com pared in a com posite draw ing (Fig. 4-8). The flu o ro g rap h ically -d etected ,
35
( S)-labeled PC -3 glycoproteins, in d icated by th e filled circles, are th e sam e
as in F ig. 4-7. The em pty circles denote the isotype p a tte rn of serum AGP as
d e te c te d by C oom assie Blue staining. AGP m ig rates as nine se p a ra te isotypes
ranging from 38,000 to 46,000 m olecular w eight and iso e le c tric points of 3.9 to
4.4. PC -3 com ponent 5, which m ig rates clo sest to AGP, has m olecular w eight
range of 35,000 to 40,000 and iso e le c tric point range of 4.1 to 4.5. A ntibodies
91
to a - l- a c id glycoprotein did not bind PC-3 com ponent 5 or any o th er acidic
PC -3 glycoprotein as shown by unsuccessful im m u n o p recip itatio n and im m uno-
a ffin ity chrom atography ex p erim ents. T herefo re, PC -3 com ponent 5 is
d istin ctly d iffe re n t from a - l- a c id glycoprotein as based on th e th re e c rite ria
of m olecular w eight range, iso e le c tric point range and an tig en icity .
92
TABLE 4-1
BPH organ cu ltu re and PC -3 cell c u ltu re bio sy n th etic
p ro d u cts th a t share iso elec tric p o in ts
and m olecular w eights:
ap p aren t
m olecular ap p aren t
BPH PC -3 w eight p i
I 1 > 2 0 0 , 0 0 0 6.0 - 7.0
m 8 52,000 - 57,000 5.8 - 6.4
IV 3 45,000 - 50,000 4.9 - 5.6
V
9 43,000 - 47,000 6.0 - 6.3
VI 6 36,000 - 40,000 5.3 - 5.5
93
TABLE 4-2
BPH and p ro s ta tic fluid com ponents th a t share
iso elec tric p o in ts and m olecular w eights:
p ro sta tic
ap p aren t
m olecular ap p aren t
BPH fluid w eight
Pi
m v n 52,000 - 57,000 5.8 - 6.4
IV n 45,000 - 50,000 5.2 - 5.7
V IV 43,000 - 47,000 5.9 - 6.4
94
TABLE 4-3
14
Im m unoprecipitations of ( C )-glucosam ine labeled glycoproteins from
su p ern atan ts of benign h y p erp lastic p ro s ta te organ cu ltu re s3:
% of to ta l dpm p re c ip ita te d
m ean ± stan d ard deviation
ex perim ent sam ple
im m une
rab b it
antibodies
serum
im m une
antibodies
non-im m une
rab b it
1 spent A nti-A G P 2 . 2 + 1.3 6.7 + 3.0
medium A nti-H p 8.9
+
5.9 6.7 + 3.0
2 spent A nti-A G P 0.7 + 0 . 1 0.9
+ 0 . 1
m edium
hom ogenate A nti-A G P 2.4 + 0.7 2.6 + 1.1
Am ount of TCA or PTA /TC A p re c ip ita b le m a te ria l used p e r experim ent
ranged from 5,800 to 26,000 dpm.
95
TABLE 4-4
14
Im m unoprecipitations o f ( C )-glucosam ine labeled glycoproteins
released into cu ltu re m edium by PC -3 cell cu ltu re,
• 3 .
and co n tro l im m unoprecipitations :
% of to ta l ra d io activ e
co u n ts p re c ip ita te d ,
m ean ± standard deviation
ex perim ent sam ple
im m une
rab b it
antibodies
im m une
antibodies
non-im m une
rab b it serum
1 spent
m edium
A nti-H p
A nti-A G P
A nti-P I
A nti-A chy
0.9 + 0.9
0.9 + 0.5
0.6 + 1.5
3.6 + 1.1
1.5 + 0.8
2 . 1 + 1 . 1
1.3 + 0.7
4.2 + 2.4
2 spent
m edium
A nti-A G P 7.8 + 0.4 7.4 + 0.2
3 (1 2 5 i ) - a g p
co n tro l A nti-A G P 70 + 1.3 4.4 + 0.5
4 (1 2 5 I)-AGP
co n tro l A nti-A G P 79 + 0.7 2.0 + 0.7
i . 14
Am ount of TCA or PTA /TC A p re c ip ita b le counts p e r ex perim ent: ( C),
175
from 8,000 to 33,000 dpm ; ( I), from 130,000 to 200,000 cpm.
96
14
Fig. 4-1. The reso lu tio n of ( C )-glucosam ine lab eled glycoproteins released
from BPH organ cu ltu re by tw o dim ensional gel electro p h o resis. R ad io activ e
com ponents w ere visualized by fluorography. The pH range fo r iso e le c tric
focusing w as 5 to 7.
14
Fig. 4-2. C om posite m ap of ( C )-glucosam ine lab eled glycoproteins released
from BPH organ cu ltu res and resolved by tw o dim ensional gel electro p h o resis.
R ad io activ e com ponents w ere d e te c te d by fluorography. Individual glycopro
tein groups are lab eled I to IX. The position of n o n -rad io activ e album in,
(ALB), is denoted by th e dashed line.
97
IEF
molecular
weight
cs
<
Q.
I
C O
Q
C O
7
1
- 9 4 kd
-68 kd
-4 3 kd
I I
-30 kd
Figure 4-1
4.5
II
BPH COM POSITE MAP
APPARENT pi
5.0 5.5 6.0 6.5 7.0
3ZIII ALB ,
rn
. . . . 2
VJ • • •
• • • t
• t •
m
IX
APPARENT
MOLECULAR
WEIGHT
-94,000
- 68,000
-43,000
-30,000
Figure 4-2
98
14
Fig. 4-3. The reso lu tio n of ( C )-glucosam ine lab eled glycoproteins released
from th e p ro sta te carcinom a cell line PC -3 by tw o dim ensional gel electro p h o
resis. C om ponents w ere d e te c te d by fluorography. The pH range for
iso e le c tric focusing was 4 to 6 . F or m eaning of arrow s, see te x t.
14
Fig. 4-4. C om posite m ap of ( C )-glucosam ine lab eled glycoproteins released
from cu ltu red PC -3 cells resolved by two dim ensional gel electro p h o resis, and
visualized by fluorography. Individual com ponents are lab eled 1 to 11. ALB,
album in.
99
SDS-PAGE
IEF
Figure 4 - 3
P C - 3 C O M P O S I T E M A P
A P P A R E N T pi
4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5
ALB
• • • • 9
• • •
Figure 4-4
molecular
weight
- 9 4 kd
- 6 8 kd
- 4 3 kd
- 3 0 kd
A P P A R E N T
M O L E C U L A R
W E I G H T
- 9 4 , 0 0 0
- 68,000
- 4 3 , 0 0 0
- 3 0 , 0 0 0
100
Fig. 4-5. Two dim ensional gel of serum , using pH range 4 to 6 in the
iso e le c tric focusing dim ension. P ro tein s w ere visualized w ith C oom assie Blue
stain . AGP, a - l- a c id glycoprotein; Achy, a -l-a n tic h y m o try p sin ; PI, a -1 -
pro tein ase inhibitor; Hp, haptoglobin; ALB, album in.
Fig. 4-6. Two dim ensional gel electro p h o resis resolving the acidic glycopro
tein s released by PC -3 cells. C om ponents w ere m etab o lically radiolabeled
14
w ith ( C )-glucosam ine and w ere d e te c te d by fluorography. The pH range was
3.8 to 5.
101
MOLECULAR
WEIGHT
68,000
43,000
«p75
pi: 3.80
g p42
pi: 4.10
4.25
4.40
4.45
4.52
-A
*
Figure 4 - 6
IEF
a
£
l
CO
o
CO
AGP
Achy
molecular
weight
Figure 4 - 5
- 9 4 kd
- 6 8 kd
p i . •
- 4 3 kd
- 3 0 kd
102
Fig. 4-7. The im proved reso lu tio n of acidic glycoproteins released by PC-3
cells in cu ltu re. These com ponents w ere m etab o lically rad io lab eled w ith
35
( S)-m ethionine and d e te c te d by fluorography. The pH range for iso elec tric
focusing was 3.5 to 5.
Fig. 4-8. C om posite m ap com paring th e 2D gel reso lu tio n of acidic glycopro
tein s released by cu ltu red PC -3 cells and th e 2D gel reso lu tio n of serum ot-1-
35
acid glycoprotein. The filled circles rep resen t the ( S)-m ethionine lab eled
PC -3 com ponents in Fig. 4-7 w hich w ere d e te c te d by fluorography. The open
circles rep resen t the isotypes of AGP which w ere visualized by C oom assie
Blue. The num bering schem e follow s th a t in Fig. 4 -4 .
103
I EF
CD
CO
a
co
f
#
p —
m o le c u la r
w e ig h t
i
C O
■ p *
kd
- 6 8 kd
- 4 3 kd
- 3 0 kd
Figure 4 -7
3.5
P C -3 COMPOSITE MAP
APPARENT pi
4.0 4.5 4.9
APPARENT
MOLECULAR
WEIGHT
-9 4 ,0 0 0
- 68,000
• • • •
0 0 0 Oru
IlMii
-4 3 ,0 0 0
-3 0 ,0 0 0
Figure 4 - 8
104
DISCUSSION
The d a ta re p o rte d in this ch ap ter show th a t BPH tissue and a p ro sta te
carcin o m a cell line in v itro biosynthesized glycoproteins which w ere released
into cu ltu re m edia. The BPH cu ltu res rem ain ed viable during th e labeling
period and m ain tain ed BPH m orphology (Tokes and D erm er, 1977). This
release of com ponents m ay re p re se n t sim ple tu rn -o v er and shedding of cell
su rface glycoproteins. A ltern ately , it m ay re fle c t the a ctiv e synthesis and
sec re tio n of cellular products. A ctive synthesis has been d e te c te d previously
in v itro w here ep ith elial cells w ere d eterm in ed to be th e predom inant source
of glycoprotein p ro d u cts (Tokes and D erm er, 1977).
Both BPH and PC -3 cu ltu res produced a larg e com ponent having a
m olecular w eight g re a te r th an 200,000 daltons. It is possible th a t this
com ponent is fib ro n ectin , as its size is consistent w ith th a t of the fib ro n ectin
subunit (230,000 daltons), w hich is known to be produced by both ra t and
hum an ep ith elial and carcin o m a cells in v itro (Neri, R uoslahti and N icolson,
1981; T ay lo r-P ap ad im itrio u , B urchell and H urst, 1981). The acidic com ponents
th a t rem ain as an unresolved stre a k on 2D gels m ay be frag m en ts of acidic
proteoglycans or glycosam inoglycans. These have been shown to be se c re te d
by m ouse p ro s ta te organ cu ltures (Sato and G yorkey, 1980) and the hum an
cu ltu res studied h e re m ay be producing sim ilar m a te ria ls. C u ltu res of BPH
and PC-3 also re le ase 7 to 9 se p a ra te glycoprotein groups th a t are w ell
resolved on 2D gels. A review of th e re c e n t lite ra tu re has not rev ealed
previous re p o rts of sim ilar m olecules produced by p ro sta te organ or cell
cu ltu res, w ith th e possible excep tio n of p ro sta tic acid phosphatase (Choe and
R ose, 1982). This m olecule has a subunit m olecular w eight range of 45,000 to
105
50,000 daltons and pi ran g e of ap p ro x im ately 5.0 to 6.0 in th e p resen ce of
u rea. These values are co nsistent w ith those found for PC -3 com ponent 3 and
BPH com ponent IV. H ow ever, u n til an im m unological analysis is m ade on
th ese tw o com ponents to d eterm in e any an tig en ic cro ssre a ctiv ity w ith pros
ta tic acid phosphatase, th e ir te n ta tiv e id en tificatio n as subunits of th is
enzym e rem ains unconfirm ed. The id e n titie s and functions of the o th er
bio sy n th etic products described h ere are unknown and aw ait fu rth e r study.
Two of the glycoproteins produced by b o th BPH and PC-3 cu ltu res
m ig rated to positions on 2D gels sim ilar to those of acu te phase p ro tein s AGP
and haptoglobin. The co n cen tratio n s of a cu te phase p ro tein s, w hich are
produced in liv er, rise in se ra of p a tie n ts w ith trau m a or various diseases,
including neoplasia (Koj, 1974). A dditionally, th e a cu te phase p ro tein s hapto
globin and a - l- a c id glycoprotein, as w ell as o th ers, w ere discovered in
p ro s ta tic fluid from h y p erp lastic tissue specim ens (C hapter II). T h erefo re, the
p ossibility ex isted th a t n eo p lastic p ro sta te tissue or cells w ere synthesizing
a c u te phase p ro tein s and co n trib u tin g to e lev ated levels of th ese p ro tein s in
serum . A lthough control exp erim en ts showed th a t radiolabeled antigen could
be successfully im m u n o p recip itated , antibodies to a - 1 -an tich y m o try p sin , a - 1 -
p ro tein ase in hibitor, haptoglobin and a - l- a c id glycoprotein failed to im m uno-
p re c ip ita te any bio sy n th etic products of BPH or PC-3 cu ltu res. Im m unoaffi-
n ity chrom atography using antibodies to AGP was sim ilarly unsuccessful in
35
binding ( S )-m ethionine-labeled glycoprotein products of PC-3 cells, even
though th e im m unoaffinity colum n successfully bound a - l- a c id glycoprotein
from serum . The conclusion is th a t BPH organ cu ltures and PC-3 c ell cu ltu res
do not produce or release com ponents th a t can be an tig en ically id e n tifie d as
serum p ro tein s.
106
In terestin g ly , w hen th e m olecular w eight and iso e le c tric points of the
bio sy n th etic p ro d u cts of PC -3 cell cu ltu re and BPH organ cu ltu re w ere
com pared, five com ponents w ere found to share th ese c h a ra c te ristic s, sugges
ting th a t th e sam e glycoproteins are produced by both cu ltu re system s. W hat
is m ore in triguing is th a t th re e of th ese five glycoproteins shared th e sam e
p ro p erties of iso e le c tric points and m olecular w eights w ith th re e p ro sta tic
fluid glycoproteins. These th re e p ro sta tic fluid com ponents w ere shown by
W estern b lo ttin g to be im m unologic ally u n related to serum p ro tein s (C hapter
II), and th e re fo re possibly unique to p ro sta tic fluid. The finding th a t
b io sy n th etic glycoproteins released from p ro sta tic organ and cell cu ltu res
share physical c h a ra c te ristic s w ith glycoproteins found in p ro sta tic fluid, along
w ith the known se c re to ry n a tu re of the p ro sta te gland (A um uller and A dler,
1979), suggests th a t th ese b io sy n th etic com ponents a re p ro s ta te ep ithelial
products w hich are p laced into glandular lum ina and are subsequently found in
p ro sta tic fluid.
Benign h y p erp lastic p ro sta te organ cu ltures and PC -3 cell cu ltu res also
produced glycoproteins in vitro th a t had very d ifferen t pi’s and m olecular
w eights when com paring th e products of the tw o cu ltu re system s. This is not
u n expected since th e BPH organ cu ltu res contained in ta c t tissue having
fibrom uscular stro m al elem en ts not p resen t in the PC-3 c ell cu ltu re. Con
versely, PC -3 cells, ad ap ted to m onolayer cu ltu re, m ay produce som e com po
nen ts m ore im p o rtan t to survival in v itro th an to m ain ten an ce of a carcinom a
cell type in vivo.
N ev erth eless, five ap p aren tly id en tical p ro sta te -a sso c ia te d glycoproteins
w ere found to be synthesized by both re la tiv e ly norm al h y p erp lastic p ro sta te
tissue as well as by a carcin o m a cell line derived from th e sam e organ. By
107
im plication, according to the hypothesis outlined in the Intro d u ctio n , if th e
biosynthesis of any of th ese glycoproteins is m ain tain ed by p ro sta te carcin o m a
cells in vivo, th e deranged carcinom a tissue stru c tu re m ay allow leakage of
th ese glycoproteins into serum . T h erefo re, th e se five glycoproteins, and
especially th e th re e th a t are sim ilar to p ro sta tic fluid com ponents, a re prim e
candidates to becom e p ro sta te m arkers.
108
CHAPTER V
SERUM a - 1-ACID GLYCOPROTEIN. ANALYSIS OF ISOTYPE HETERO
GENEITY IN NORMAL AND DISEASE STATES BY TWO DIMENSIONAL GEL
ELECTROPHORESIS.
109
INTRODUCTION
a -l-A c id glycoprotein (AGP) is a m ajor co n stitu en t of norm al hum an
serum . The co n cen tratio n of this a cu te phase re a c ta n t rises in m any
conditions of tissue dam age or d eg en eratio n such as in flam m ation and neo
p lasia (Koj, 1974; K indm ark, 1976). This rise o fte n c o rre la te s w ith tum or
burden (Koj, 1974; C ooper and Stone, 1979). The co m plete am ino acid
sequence of AGP has been d eterm in ed and the carb o h y d rate stru c tu re s have
been ex ten siv ely in v estig ated (Schmid e t al., 1973; F o u rn et e t al., 1978).
Am ino acid su b stitu tio n s have been observed in 21 of 181 positions. AGP
consists of 45% carb o h y d rate, 11% of w hich is N -acety l neuram inic acid
(NANA) positioned term in ally on the polysaccharide side chains (see review ,
Schmid, 1975). The carb o h y d rate m o iety is com posed of five hetero p o ly sac
charide units linked N -glycosidically to asparagine residues. M icrohetero
g en eity of AGP was d em o n strated using various e lec tro p h o retic and iso e le c tric
focusing techniques (Schmid e t al., 1962; Rudm an e t al., 1972; A ltland e t al.,
1982; Gordon and D ykes, 1972; T am ura e t al., 1981; N icollet e t al., 1981).
H ow ever, p recise q u a n tita tio n of th e various isotypes in norm al and p a tie n t
se ra has n o t been perform ed.
The w ork described h ere shows th a t a - l- a c id glycoprotein from norm al
serum can be co n sisten tly resolved in to a group of a t le a st nine spots, or
isotypes, by tw o dim ensional gel electro p h o resis. The isotypes display a wide
range of values fo r both iso e le c tric points and m o lecular w eights. The
ap p aren t iso e le c tric points, m olecular w eights and re la tiv e distrib u tio n of
isotypes are rep o rted . A dditionally, th e 2D gel p a tte rn s of AGP isotypes in
110
th e sera of p a tie n ts w ith in flam m ato ry and neo p lastic conditions are shown to
d iffer from norm al.
I l l
RESULTS
a -l-A c id glycoprotein m ig rated to the c e n te r of gels when serum was
su b jected to 2D electro p h o resis using pH range 3.5 to 5. U nder th e se
conditions, AGP ap p eared as the m ajor serum com ponent and was co n sisten tly
resolved in to d istin c t isotypes as visualized by th re e d iffe re n t staining te c h
niques: C oom assie Blue, periodic acid-S chiff's re a c tio n (PAS) and tw o types of
silver stain (Fig. 5-1). In every norm al serum exam ined, nine c h a ra c te ristic
isotypes w ere recognized w ith C oom assie Blue and PAS. A te n th , m ore basic
isotype was d e te c te d in tw o of four sera by silver stain w hich, as discussed
below , has g re a te r sen sitiv ity than C oom assie Blue. All the isotypes visualized
by C oom assie Blue w ere im m unologically id en tified as AGP on W estern blots
tre a te d w ith anti-A G P antibodies and rad io io d in ated p ro te in A. This tech
nique, w hich is a t le a st 1 0 tim es m ore sensitive th an silver stain , d e te c te d no
additional isotypes in norm al serum (Fig. 2-11).
The distrib u tio n of norm al serum AGP isotypes to g e th e r w ith th e ir
m olecular w eights and iso e le c tric points are sum m arized in T able 5-1. The
nine m ajor isotypes ranged in th e ir m olecular w eights from 39,600 to 48,200
daltons. The sm allest m olecular w eight d ifferen ce, 700 daltons, was seen
b etw een isotypes 5 and 6 . The larg est in crem en t, 1500 daltons, was b etw een
isotypes 2 and 3. All th e o th er d ifferen ces averaged approxim ately 1000
daltons. The iso e le c tric points varied from 3.93 to 4.37. The la rg e st
d ifferen ces w ere observed b etw een isotypes 3 and 4, 0.09 pH u n its, and
b etw een isotypes 6 and 7, 0.07 pH units. The sm allest in crem en t, 0.03 pH
u n its, was seen b etw een isotypes 5 and 6 . The o th e r in crem en ts averaged 0.05
pH units.
112
The re la tiv e d istrib u tio n of p ro tein am ong various isotypes was estim a
te d by m easuring th e op tical density of the spots stain ed w ith C oom assie Blue.
Since th e absolute values of th e o p tical density showed considerable v ariatio n
due to staining, d estain in g and gel drying conditions, th e q u a n tita tio n of the
various isotypes is expressed as a p ercen tag e of th e to ta l o p tical absorption.
For the purpose of e stim atin g th e isotype distribution, id en tical m olar ex tin c
tion co efficien ts w ere assum ed for all com ponents. The m ajor com ponent w as
isotype 5, rep resen tin g approxim ately one fo u rth of the to ta l serum AGP. The
re la tiv e am ounts of th e m ore acidic and m ore basic iso ty p es gradually de
creased to approxim ately 2% of the to ta l p ro tein . Id en tical d istrib u tio n of
isotypes was ob tain ed w hen an AGP sam ple purified from pooled, norm al
serum was analyzed. U sing th e m ean value of 900 yg AGP p er ml of norm al
serum , as d eterm ined by n ep h elo m etry , th e co n cen tratio n s of various isotypes
in the serum can be e stim a te d (Table 5-1). The co n cen tratio n s ranged from 14
to 249 ]ig per ml of serum . Since 30 pi of serum was analyzed in each assay,
0.2 yg was the sm allest am ount of isotype d e te c ta b le . The o p tical density of
C oom assie B lue-stained spots w hich contained less th an this am ount of p ro tein
could not be a cc u ra te ly m easured. A lthough sm aller am ounts can be visua
lized by silver stain , th e rep ro d u cib ility of th is technique was in su fficien t to
allow consistent q u a n tita tio n of isotype distribution.
A single sam ple of norm al serum was re p e ate d ly su b jected to electro p h o
resis and the am ounts of p ro te in per isotype w ere d eterm in ed to establish the
rep ro d u cib ility of th is system . The m eans and stan d ard deviations of th ese
values are liste d in T able 5-2. The c o effic ien t of v ariatio n b etw een re p lic a te
d eterm in atio n s of isotype d istribution ranged from 5% for isotype 5, w hich had
highest optical density, to 25% fo r isotype 9 » w hich had low est op tical density.
113
The m olecular w eights and iso e le c tric points of isotypes from th e single
sam ple w ere indistinguishable from th e m ean values obtained from th e te n
norm al individuals.
P u rified AGP from pooled norm al serum was resolved on tw o sep a ra te
2D gels. One was stain ed w ith C oom assie Blue to visualize p ro tein s, w hile th e
o th er w as stain ed fo r carbohydrates by th e PAS re a ctio n . O ptical densito
m e try w as p erfo rm ed on the gels and th e ra tio of adsorption of PAS stain to
C oom assie Blue stain was d eterm in ed fo r each isotype. The m ore acidic
isotypes, 2, 3 and 4, had a m ean ra tio value of 0.42, w hereas th e m ore basic
isotypes, 7, 8 and 9, had a m ean ra tio value of 0.29. These d a ta show th a t
m ore in ten se carb o h y d rate staining w as observed in th e acidic isotypes th an in
th e basic ones, w hich establishes th a t th e isotype carb o h y d rate co n ten t
in creases w ith d ecreasing pi.
AGP was p a rtia lly p u rified from serum by tre a tm e n t w ith 0.6 JN
p erch lo ric acid w hich p re c ip ita te s m ost p ro tein s but does not insoluabilize
AGP or a lte r its isotype m ig ratio n p a tte rn . This AGP p re p a ratio n was tre a te d
w ith neuram inidase from C lostridium p erfringens to en zy m atically rem ove th e
n eg ativ ely charged sialic acid m o ieties, N -acety l neuram inic acid (NANA).
The resu ltin g asialo-A G P was ru n on 2D gels using e ith e r 3.5 to 5, or 5 to 7 pH
ranges. A sialo-A G P did not en ter the low pH range 2D gel, but ra th e r resolved
on th e high pH range gel as five d istin ct spots having pi’s from 5.2 to 5.6 and
m olecular w eights of 42,000 or 43,000 daltons (Fig. 5-2). The p ro tein group
seen at 68,000 m o lecu lar w eight is th e neuram inidase enzym e. N euram inidase
tre a tm e n t is ap p aren tly com plete since addition of m ore enzym e to th e asialo-
AGP p rep aratio n did not change pi values any fu rth er. H ow ever, such
en zy m atic tre a tm e n t usually does not rem ove all sialic acid m oieties from
1x4
glycoproteins (Gordon and D ykes, 1972). A sialo-A G P was not antig en ically
a lte re d as it form ed a p re c ip itin arc on double im m unodiffusion w ith rab b it
antibodies to n ativ e AGP. These resu lts confirm th a t NANA is the m ajor
cause of th e acidic pi range of th e u n tre a te d m olecules (Schmid, 1975). Also,
th e n eg ativ ely charged NANA m olecules are responsible fo r th e ap p aren t wide
variations in m olecular w eight of n a tiv e isotypes since asialo-A G P displayed a
m uch n arrow er m olecular w eight range. The iso e le c tric point h etero g en eity
was not to ta lly elim inated by neuram inidase tre a tm e n t, in d icatin g th a t e ith e r
som e NANA m oieties rem ain a tta c h e d or th a t o th er charge d ifferen ces still
exist b etw een asialo-A G P m olecules.
Two dim ensional try p tic pep tid e analysis was used in a tte m p ts to d e te r
m ine w h eth er isotype pi d ifferen ces w ere due in p a rt to am ino acid d iffe r
ences. N ative or asialo-isotypes w ere individually sliced from 2D gels and
125
rad ioiodinated w hile rem aining w ithin th e gel m atrix . The ( I)-labeled
glycoproteins w ere cleaved by p e rm ittin g trypsin to diffuse in to the gel slices
and allow ing th e re su lta n t try p tic p eptides to diffuse out. R adioiodinated
pep tid es w ere individually applied to square thin lay er cellulose p la te s and
su b jected to electro p h o resis in th e first dim ension. Subsequently, th e air-d ried
p la te s w ere developed in th e second dim ension by ascending chrom atography
and th e sep a ra te d p ep tid es w ere d e te c te d by autoradiography. When n ativ e
AGP isotypes 4 and 7, and all five asialo-A G P isotypes w ere analyzed, all
try p tic p ep tid e m aps displayed 25 spots (Fig. 5-3). W ithin th e reso lu tio n lim its
of this system , all autoradiograph p a tte rn s w ere indistinguishable. These
re su lts show th a t th e causes of pi d ifferen ces b etw een isotypes cannot be
discerned by pep tid e analyses and th a t the absence of NANA has no e ffe c t on
try p tic p ep tid e m igration in th is system .
115
Since a - l- a c id glycoprotein was found in p ro sta tic fluid (C hapter n) and
since acidic glycoproteins w ere found to be synthesized by p ro sta tic cell and
organ cu ltu res (C hapter IV), it seem ed ap p ro p riate to lo calize AGP antigens in
p ro sta te tissue sectio n s by im m unohistochem ical staining. AGP was p u rified
from norm al serum by p erch lo ric acid p re c ip ita tio n and Sephadex G-100
chrom atography, and used to im m unize a rab b it. The an tiseru m , w hich form ed
a p recip itin arc on double im m unodiffusion versus serum , was fra c tio n a te d
w ith am m onium su lfa te. The im m unoglobulins w ere subsequently rim into an
im m unoaffinity colum n consisting of AGP im m obilized to Sepharose 4B. Spe
cifically bound anti-A G P im m unoglobulins w ere elu ted w ith th e ch aotropic
agent 3M potassium th io cy an ate and dialyzed extensively against phosphate
b u ffered saline. Both this antibody p rep aratio n and com m ercially available
anti-A G P im m unoglobulins w ere used fo r th re e lay er peroxidase antiperoxidase
staining of fo rm alin -fix ed , p araffin-em bedded p ro sta te tissu e sectio n s. W ith
bo th antibody p rep aratio n s th e cytoplasm of the glandular epithelim stain ed
positively in 7 of 7 BPH sectio n s (Fig. 5-4). In any p a rtic u la r tissue sectio n ,
not all acini stained, but w ithin positive acini all th e ep ith elial cells stain ed
positively. This ty p e of staining p a tte rn m ay be a rtifa c tu a l, resu ltin g from
uneven fix atio n of the tissue w ith form alin. G ranulocytes and blood vessels
stained p o sitively fo r AGP and occassionally som e ep ith elial su rfaces stain ed
m ore darkly th an cytoplasm . Only 4 of 8 p rim ary p ro sta te carcinom a cases
stained p o sitively fo r AGP. In stained sectio n s not all carcinom a cells w ere
positive, but ra th e r a patchy, h eterogeneous staining p a tte rn was seen. In two
of th ese cases, m e ta sta se s to lym ph nodes displayed th e sam e staining p a tte rn
of can cer cells (Fig. 5-5). In neg ativ e controls, w here p rim ary im m unoglobulin
was ab sent or was su b stitu ted by non-im m une serum , no staining was observed.
116
T h erefo re, AGP or an A G P-like antigen is p resen t in p ro sta te epithelium but
this antigen's expression is not n ecessarily m ain tain ed in carcin o m a cells.
An analysis o f AGP isotype d istrib u tio n was m ade in p a tie n ts w ith
in flam m ato ry and benign hyp erp lastic conditions, and in p a tie n ts w ith p ro sta te
and b re a st carcinom a. In Fig. 5-6, th e d istribution of AGP isotypes in th e
serum of an advanced case of p ro sta te carcinom a is d em o n strated on a
C oom assie B lue-stained 2D gel and com pared to th e p a tte rn from norm al
serum . In addition to the c h a ra c te ristic , re la tiv e ly acidic isotypes 1 through 4,
a m ore acidic com ponent, isotype 0, becam e visible. The m ore b asic isotypes,
8 and 9> d ecreased to a level w here th ey w ere not d e te c ta b le . Isotype 0 was
observed in th e p reo p erativ e se ra of 1 of 7 benign p ro sta te hyperplasia
p a tie n ts, 1 of 5 p ro s ta te can cer (stages B and C) p a tie n ts, and 3 of 3 b re a st
in tra d u c ta l carcinom a p a tie n ts. It was also d e te c te d in 2 of 2 rheu m ato id
a rth ritis and 2 of 3 advanced p ro sta te cancer cases w ith d istan t m e ta sta ses.
In addition, one advanced CAP serum contained an even m ore acidic compo
nent (pi 3.82) th an isotype 0, as rev ealed only by silver staining.
In Table 5-3, th e p e rc e n t distributions of AGP isotypes are sum m arized
for the sera of p a tie n ts w ith rheum atoid a rth ritis, and p reo p erativ e sam ples
from cases of benign p ro sta te hyperplasia, p ro sta te and b re a st carcinom as. In
th ese p a tie n ts, a sig n ifican t sh ift appears tow ard g re a te r p erc en ta g e of
p ro tein in th e m ore acidic isotypes (0 through 4). T here is a co n cu rren t
d ecrease in p ro tein in the m ore basic isotypes (5 to 9)* The significance of the
acidic sh ift was calcu lated by d eterm ining th e ra tio :
Z (percentage values, isotypes 0 through 4)
£ (percentage values, isotypes 5 through 9)
117
F o r norm al se ra th is ra tio , nam ed th e isotype ra tio , had a m ean value of 0.74
w ith stan d ard dev iatio n 0.21. No individual norm al serum had a value above
1.0. The ra tio values from acid -sh ifted p a tie n t se ra ranged from 1.11 to 2.73
w ith a m ean of 1.51. These e lev ated isotypes ra tio s w ere d eterm in ed to be
significantly d iffe re n t from norm al sera ra tio s a t a 95% confidence level using
th e stu d en t's t te st. D em onstrable acidic sh ift was observed in all p a tie n t sera
ex cep t 1 of 3 b re a st can cer p a tie n ts and 1 of 2 rh eu m ato id a rth ritis cases.
A lthough th e AGP in only one a rth ritic case ap p eared acid -sh ifted , both
a rth ritis p a tie n ts' sera con tain ed isotype 0 and b o th had e lev ated AGP levels.
All BPH p a tie n t sera w ere acid -sh ifted , b u t only 3 of 7 had significant
elevations of AGP. In one b re a st can cer and tw o p ro sta te can cer p a tie n ts, a
significant acidic sh ift was observed even though th e to ta l serum AGP
co n cen tratio n was w ithin th e norm al range. C onversely, th e one b re a st can cer
serum which failed to show an acidic sh ift contained th e elev a te d co n cen tra
tion of 2000 yg of AGP p er ml of serum . T h erefo re, th e conclusion is th a t th e
p resence of isotype 0 or an acidic sh ift is independent of to ta l AGP co n cen tra
tion in serum .
An illu stra tio n of th e p e rc en t distrib u tio n of AGP isotypes in norm al,
benign p ro sta te h yperplasia and advanced, m e ta s ta tic p ro s ta te carcinom a
p a tie n ts is p resen ted in F ig. 5-7. This graph once again d em o n strates the sh ift
in p ro tein distrib u tio n tow ards acidic isotypes. An even m ore su b stan tial
acidic sh ift is seen in sera of p a tie n ts w ith advanced, m e ta s ta tic p ro sta te
carcinom a th an in th e se ra of p reo p erativ e p a tie n ts sum m arized in T able 5-3.
The isotype containing th e g re a te s t p e rc en ta g e of p ro te in changed from
isotype 5 to isotype 3. The m ean isotypes ra tio for th ese th re e p a tie n ts w as
2.65. The highest levels of isotype 0 are also seen in th ese cases.
118
TABLE 5-1
C h a ra c te ristic s o f norm al serum a - l- a c id glycoprotein
isotypes resolved on 2D gels .
AGP
iso types
ap p aren t
m olecular
w eight
ap p aren t
iso electric
point
% of to ta l
AGP p e^
iso type
co n cen tratio n
M>g/ml serum
1 48,200+3,200 3.93+0.12 3 .2 + 1 .2 29
2 47,100+2,900 3.97+0.13 7 .7 + 1 .7 69
3 45,600+3,100 4.02+0.13 1 2.2+ 2.3 1 1 0
4 44,500+2,600 4.11+0.12 1 7 .2 + 2 .4 155
5 43,500+2,400 4.15+0.11 2 7 .7 + 1 .9 249
6 42,800+2,400 4.18+0.12 18.8+ 3.1 169
7 41,900+2,300 4.25+0.13 7 .7 + 1 .1 69
8 35,800+2,000 4.31+0.12 3 .9 + 1 .4 35
9 39,600+1,800 4.37+0.12 1 .6 + 0 .9 14
Values are based on ten d iffe re n t sera from h ealth y donors (m ean + stan d ard
deviation).
V alues w ere determ in ed from C oom assie B lue-stained gels.
Cm
The m ean co n cen tratio n of AGP in the serum o f h ealth y persons is 900
Hg/ml.
119
TABLE 5-2
R eproducibility in determ ining
th e p e rc e n ta g e s of AGP iso ty p es .
AGP P e rc e n t of to ta l C o efficien t
iso ty p e AGP p e r isotype of v ariatio n
1 2 . 8 + 0.5 18
2 7 .6 + 1 . 1 14
3 12.3 + 1 . 6 13
4 15.8 +
1.9 1 2
5 2 6 . 8 + 1.3 5
6 19.7 + 1.4 7
7 8 . 0 +
0.9 11
8 4 .7 + 0 . 8 17
9 2 . 0 + 0.5 25
Values a re based on six d iffe re n t 2D gel sep aratio n s o f th e sam e norm al serum
sam ple (m ean + standard deviation).
120
TABLE 5-3
b c
P ercent distribution of AGP isotypes. ’
rheum atoid
AGP norm al arth ritis BPH CAP CAB
iso types sera (n=1 0 ) sera (n=2 ) sera (n=7) sera (n=5) sera (n=3)
0
a
0 . 9 + 0 . 6 0.1 + 0.3 0.8 + 1.7 1.2 + 0.7
1 3 .2 + 1.2 3 .7 + 1.6 6.3 + 2.7 7.3 + 2.2 4 .9 + 2 . 1
2 7 .7 + 1.7 9 . 8 + 2 . 1 15.6 + 2.9 15.3 + 2.5 12.5 + 4.7
3 12.2 + 2.3 14.6 + 1.4 18.3 + 2.0 15.9 + 1.4 15.4 + 2.9
4 17.1 + 2.4 22.7 + 0.2 19.9 + 2.3 20.8 + 4 .2 20.9 + 0.3
5 27.7 + 1.9 23.8 + 0.5 22.5 + 3 .2 22.9 + 2 . 6 23.2 + 2.4
6 18.7 + 3.1 15.1 + 1.6 11.6 + 3.0 10.8 + 3 .2 13.7 + 3 .2
7 7 .7 + 1.1 6.1 + 1.3 3 .6 + 1.5 4 .2 + 1.0 5.5 + 2.2
8 3 .9 + 1.4 2 .6 + 1.3 1.8 + 0.5 1.9 + 0.4 1.9 + 1.9
9 1.5 + 0.9 0.9 + 0 . 6 0.5 + 0.5 0 .2 + 0.4 0.9 + 0.9
g
^Below the level of d etection using Coomassie Blue stain for protein.
Serum sam ples were collected preoperatively.
i-*
IN)
Values are mean + standard deviation. For each column, n denotes the num ber of sera examined.
I — *
Fig. 5-1. Two dimensioned gel reso lu tio n of a - l- a c id glycoprotein from serum
of a health y , norm al volunteer. The pH range was 3.5 to 5 and p ro tein was
visualized w ith C oom assie Blue. N um bers 1 through 9 re p re se n t individual
AGP isotypes.
Fig. 5-2. Two dimensioned gel of a - l- a c id glycoprotein tre a te d w ith neura
m inidase. A pH range 5 to 7 was u tilized and p ro tein s w ere stain ed w ith
C oom assie Blue. L e tte rs A through E den o te individual asialo-A G P isotypes.
122
ACIDIC
CD
<
Q _
I
CO
o
CO
IEF
BASIC
molecular
weight
(kilodaltons)
-66 kd
-45 kd
Figure 5-1
-30 kd
IEF
A BC DE
a *
molecular
w eight
68 kd
43 kd
- 3 0 kd
Figure 5-2
123
Fig. 5-3. A utoradiographs of tw o dim ensional try p tic pep tid e m aps. Individual
AGP isotypes w ere cu t from 2D electrophoresis gels said used fo r try p tic
pep tid e analysis (see M aterials and M ethods). F irst dim ension p roceeded from
th e origin to th e top. Second dim ension proceeded from le ft to rig h t. L e ft
panels sure th e autoradiographs and rig h t panels sore sk etch es clarifying the
positions of th e try p tic pep tid es. Isotype 7 was from n a tiv e , u n tre a te d AGP.
A sialo-A G P isotype D wsus obtained a fte r neuram inidase tre a tm e n t (Fig. 5-2).
Solid c ircles re p re se n t d ark est spots on autoradiographs. D ashed circles
rep re se n t fain t spots.
124
electrophoresis
I
»
chromatography-
1
I
Figure 5-3
1
O
O
o
0
o
O ’
0 '
C: O
O c
o
o o
o
0
O
AGP
isotype
7
o
origin
0
0
0
0
o
0
Or-
: 0 °
O
0
• > 0
o
0
0 0
0 o
asialo-AGP
isotype
D
40
Fig. 5-4. T hree layer peroxidase antiperoxidase staining of form alin fixed
BPH tissue sectio n using anti-A G P antibodies. H em atoxylin was used as
co u n terstain . M agnification was 100X.
Fig. 5-5. T hree lay er peroxidase antiperoxidase staining of a form alin fixed
lym ph node sectio n containing m e ta sta tic p ro sta te carcinom a. A nti-A G P
antibodies w ere used and th e sectio n was co u n terstain ed w ith hem atoxylin.
M agnification was 250X.
126
Figure 5~4
*
L>>
& r y $ £ )
< • ■ * * !S j v * V ;
-Jk
*
; > *
%
»•. •■ * * $
* . t - j ? e >
.1% % T1
■ i
n
*■
*
w -
N * V >
J *
» v *
«5I
SfcfV
• 2 v.
4 $
/ > •
1 l ” A .
r • r r •4
f e
. *9*
Fig. 5-6. Two dim ensional gels of serum , pH ran g e 3.5 to 5. The upper panel
dep icts AGP isotypes resolved from norm al serum . The low er panel illu stra te s
th e a cid -sh ifted d istrib u tio n of AGP isotypes obtained from th e serum of a
p a tie n t w ith advanced, m e ta s ta tic p ro sta te carcinom a.
128
ACIDIC
C D
<
O-
I
C O
o
co
IEF
-f?
3 4 5 J } I
BASIC
8 9
molecular
weight
(kilodaltons)
-66 kd
- 4 5 kd
-30 kd
1 in f M
2 3 4 5 6
66 kd
- 4 5 kd
-30 kd
Figure 5 -6
129
Advanced
Normal BPH CAP
(n =10) (n = 7) (n = 3)
□ □ ■
2 0
Isotypes
i1 f a
Figure 5-7
Fig. 5-7. Bar graph illu stra tin g th e re la tiv e distrib u tio n of AGP p ro tein am ong
th e isotypes in norm al individuals, p re-o p erativ e benign p ro sta te hyperplasia
(BPH) and advanced m e ta s ta tic p ro sta te carcin o m a (CAP) p a tie n ts.
130
DISCUSSION
a -l-A c id glycoprotein from norm al serum was co n sisten tly resolved into
a t le a st 9 subgroups on 2D gels using pH range 3.5 to 5. M icro h etero g en eity
was first noted by Schm id w hen pure AGP was resolved in to seven p ro tein
bands by starch gel electro p h o resis a t pH 3 (Schmid e t al., 1962). M ore
re c en tly , sev eral o th er in v estig ato rs have also no ted sim ilar m icro h etero
g en eity as d em o n strated by one dim ensional gel electro p h o resis (Rudman e t
al., 1972; A ltland e t al., 1982) and by iso elec tric focusing (Gordon and D ykes,
1972; T am ura e t al., 1981; N icollet et al., 1981). The 2D gel system used in
this study enables the sim ultaneous d eterm in atio n of iso elec tric points and
m olecular w eights as w ell as th e isotype distribution.
Serum co n ce n tra tio n of the m ost prom inent com ponent, isotype 5, was
249 lig/m l. D ecreasing am ounts of p ro tein w ere p re sen t in th e m ore acidic
and m ore basic isotypes. The le a st prom inent isotypes d e te c te d by C oom assie
Blue staining, 1 and 9, w ere p resen t in co n cen tratio n s of 29 and 14 y g /m l,
resp ectiv ely . An additional, m ore acidic iso type has been d e te c te d in sera
from p a tie n ts w ith p ro sta te and b re a st can cer, rh eu m ato id a rth ritis and benign
p ro sta te hyperplasia. A sig n ifican t sh ift tow ards the m ore acidic isotypes was
also evident in m ost of th ese p a tie n ts. The acidic sh ift appeared to be
independent of b o th th e overall co n cen tratio n of AGP and th e presence of
isotype 0. The abnorm al p a tte rn s ap p ear to re su lt from d isproportionate
am ounts of the m ore acidic isotypes. In all cases th e a cid -sh ifted p a tte rn m ay
re fle c t an activ e process of tissue injury.
The sen sitiv ity of AGP isotype d etectio n , using C oom assie Blue stain ,
was 5 y g /m l w hen a serum sam ple volum e of 30 yl was analyzed. This sam ple
131
size affo rd ed th e b est com prom ise b etw een high p ro tein loading and optim al
AGP isotype resolution. Using silver stain, th e sen sitiv ity of p ro tein d e tec tio n
in creased to 0.5 y g /m l of serum . H ow ever, th e rep ro d u cib ility of silver stain
was not suitable fo r a cc u ra te q u an tita tio n of isotype distribution. W estern
b lo ttin g of 2D gels rep resen ts an even m ore sen sitiv e analysis. This technique
req uires th e binding of sp ecific antibodies to pro tein s which have been
e lectro p h o retic ally tra n sfe rre d to a n itro cellu lo se sh eet. Subsequently, th e
bound antibodies w ere d e te c te d w ith radioiodinated p ro tein A and autoradio
graphy. This m ethod can id en tify an e stim a te d 50 ng of p ro tein p er ml of
serum . U sing C oom assie blue and silver stain techniques, th e m ost acidic
com ponent, isotype 0 , was below th e level of d e tec tio n in all norm al sera,
indicating th a t its co n cen tratio n was less than 0.5 yg /m l. F u rth erm o re, in the
two norm al se ra studied by W estern b lo ttin g , this com ponent was also below
th e level of d e te c tio n suggesting th a t if p resen t, it m ust have a co n cen tratio n
of less th an 50 ng /m l. H ow ever, isotype 0 was d e te c te d by C oom assie Blue in
9 out of 2 0 se ra of p a tie n ts analyzed in this study, a t co n cen tratio n s e stim ate d
to range from 1 to 70 yg /m l. A dditionally, in one specim en from a p a tie n t
w ith advanced, m e ta sta tic p ro sta te can cer, th e p resence of an even m ore
acidic isotype was observed a t pi 3.82, having an e stim ate d co n cen tratio n of
0.5 y g /m l. The presence of ex tra, acidic isotypes did not c o rre la te w ith
o v erall AGP co n cen tratio n in serum . The m icro h etero g en eity p a tte rn ob
served by A ltland e t al. (1982) w as also independent of the co n cen tratio n of
AGP in th e plasm a. A t this tim e it is u n c ertain w h eth er th e presence and
co n cen tratio n of th ese m ost acidic isotypes c o rre la te w ith e x te n t of disease.
The e x a c t m olecular basis for th e variations am ong th e isotypes is not
known. All of the isotypes w ere recognized on a W estern blot by rab b it
132
antibodies to AGP. Sim ilarly, im m unoaffinity p u rified AGP displayed the
sam e isotype distrib u tio n as observed in the whole serum (C hapter IV). These
observations show th a t rab b it im m unoglobulins do not d iscrim in ate am ong th e
isotypes and suggest th a t polyclonal antibodies cannot distinguish th e m inute
an tigenic d issim ilarities th a t m ay exist b etw een isotypes. D ifferen ces in
glycosylation could cause m ost of the variations am ong isotypes. A gradual
in crease in th e carbohydrate to p ro tein ra tio is observed w ith increasingly
acidic iso e le c tric points in d icatin g th a t m ore carbohydrate is p re sen t in acidic
isotypes. Various com binations of sialic acid residues and n e u tra l carbohy
d ra te m oieties could explain in crem en tal m olecular w eight d ifferen ces be
tw een isotypes. F o r exam ple, addition of 2 NANA residues to a m olecule
could re su lt in an approxim ate m olecular w eight in crease of 700 daltons, as
w ell as increasing th e to ta l n eg ativ e charge on th e m olecule, resu ltin g in an
a lte re d pi. An a lte rn a te explanation of m olecular w eight d ifferen ces could be
th e anom alous behavior of som e glycoproteins seen in electro p h o resis due to
changes in th e SDS: glycoprotein ra tio (Frank and R odbard, 1975) which is
usually 1.4:1 fo r m ost p ro tein s (Reynolds and Tanford, 1970). V ariable
am ounts of n eg ativ ely charged NANA m o ieties on d ifferen t isotype m olecules
could a lte r th e num ber of n eg ativ ely charged SDS m olecules bound. Such
changes would re su lt in m odified e lec tro p h o retic m ig ratio n and a lte re d ap
p a re n t m o lecu lar w eight. Support for th is explanation com es from th e finding
th a t the 39,600 to 48,200 m olecular w eight range of n ativ e AGP changes to
th e narrow range of 42,000 to 43,000 daltons fo r asialo-A G P. N euram inidase
tre a tm e n t also a lte re d the iso e le c tric points of every AGP isotype confirm ing
th e ex ten siv e av ailab ility of N -acety l neuram inic acid in th e se m olecules
(Schmid, 1975). H ow ever, such enzym e tre a tm e n t failed to convert all of the
133
isotypes into a single hom ogeneous com ponent, as determ in ed in this study by
two dim ensional gel electro p h o resis and as noted by o th e r in v estig ato rs
(A ltland e t al., 1982; Gordon and Dykes, 1972). This suggests th a t ad ditional
charged m oieties, e ith e r from p ro te in backbone or from the polysaccharide
groups, co n trib u te to th e iso type h e tero g en eity . A ltern ately , neuram inidase
tre a tm e n t m ay not rem ove all of the NANA residues (Gordon and Dykes,
1972).
Two dim ensional try p tic pep tid e m ap analysis w as used to fu rth e r
in v estig ate the m olecular d ifferen c es b etw een isotypes th a t re su lt in p i
h e tero g en e ity a fte r neuram inidase tre a tm e n t. In such an analysis, the
m obility of p ep tid es in e ith e r dim ension depends p rim arily on th eir am ino acid
sequences (Moss, 1979). The first dim ension sep a ra te s p ep tid es on th e basis of
charge and the second dim ension on th e basis o f hydrophobicity. From all the
AGP subgroups exam ined, 25 d iffe re n t p ep tid e spots w ere visualized. The spot
p a tte rn s w ere indistinguishable w hether derived from asialo-A G P isotypes or
from n ativ e isotypes th a t had not been neuram inidase tre a te d . The absence of
m ajor changes on p ep tid e m aps o f d iffe re n t AGP isotypes in d icates th a t the
m ass of carb o h y d rate p re se n t on AGP does n o t g re a tly influence th e m igration
p a tte rn o f p ep tid es on try p tic p ep tid e m aps. Elder e t al. (1977a) have
obtained sim ilar findings for glycoproteins derived from m onkey leukem ia
viruses and from D ictyostelium discoideum .
The series of five try p tic p ep tid es sep arated by electro p h o resis in the
first dim ension appear to be five carb o h y d rate-co n tain in g p ep tid es. Five
glycopeptides are ex p ected since this is th e known num ber of carb o h y d rate
a tta c h m e n t site s (Schmid e t al., 1973). H ow ever, th ese 5 spots did n o t
m ig rate in th e second dim ension. E lder and cow orkers (1977b), noted th a t
134
glycopeptides do n o t m ig rate in th e apolar solvent used for th e second
dim ension of th ese try p tic pep tid e m aps. K am iyam a and Schm id (1962a,b)
no ted sim ilar findings. T h erefo re, th ese 5 p eptides are id en tified as glycopep
tides.
AGP polypeptide has a th e o re tic a l maximum of 24 d iffe re n t try p tic pep
tides. H ow ever, only 11 of th ese are ex p ected to be labeled by chloram ine T
cata ly z ed radioiodination of AGP's tyrosine residues. T here a re several
possible explanations for finding m ore radioiodinated spots th an e x p ected on
pep tid e m aps. F irst, studies by K rohn e t al., (1977) in d icate th a t histidine and
phenylalanine residues are also susceptable to radioiodination by th e chlora
m ine T m ethod. Second, th e trypsin p re p a ratio n used fo r th ese experim ents
was not tre a te d w ith th e chym otrypsin inhibitor tosylam ide phenylethyl
chlorom ethyl ketone, and co n tam in an t chym otryptic a c tiv ity m ay have cre
ated additional peptides (K ostka and C arp en te r, 1964). Finally, trypsin
digestion m ay have been incom plete considering th a t enzym e tre a tm e n t was
p erfo rm ed w ithin th e polyacrylam ide gel. Any or all of th ese fa c to rs could
explain why th e num ber of try p tic peptides th a t w ere obtained was g re a te r
th an ex p ected . O verall, th e p ep tid e m aps of th e exam ined isotypes w ere
indistinguishable, in dicating th a t this m ethod is unable to d e te c t am ino acid
d ifferen ces known to exist b etw een AGP m olecules (Schmid e t al., 1973).
H ow ever, this technique cannot d e te c t am ino acid su b stitu tio n s p resen t in the
glycopeptides since th ey w ere not resolved in th e second dim ension of p ep tid e
m apping. T h erefo re, am ino acid su b stitutions cannot be ruled out as a cause
of pi h etero g en eity .
The p rim ary synthesis of AGP occurs in the liver (Jam ieson and A shton,
1973a, 1973b). H ow ever, th e synthesis of A G P-like m olecules by leukocytes
135
has been re p o rte d (G ahm berg and Anderson, 1978). Also, AGP was found to be
p resen t in p ro sta tic fluid (C hapter IE ) and acidic glycoproteins w ere seen to be
produced by p ro sta te cell and organ cultures (C hapter IV). When stain ed w ith
th e th re e layer peroxidase antiperoxidase m ethod, AGP antig en w as found in
hyp erp lastic p ro sta te ep ith elial cells and in a portion of th e p ro sta te cancer
cells in p rim ary and m e ta s ta tic lesions. H ow ever, no evidence was found fo r
in vitro biosynthesis of AGP by BPH or PC -3 cu ltu res (C hapter IV). If cancer
or ep ith elial cells tru ly synthesize AGP or an A G P-cross re a c tiv e m olecule, a
possible reason fo r th e in ab ility to d e te c t it in cu ltu re m edia is th a t it is
re ta in ed by th e cells. Thus, th e antigen could be m em brane bound or enclosed
in a m em brane bounded co m p artm en t. AGP was found to be m em brane
asso ciated on th e su rface of leukocytes (Gahm berg and A nderson, 1978). If it
is sim ilarly m em brane asso ciated in p ro sta te ep ith elial cells, it would likely be
unavailable as a w a te r soluble com ponent and would not be d e te c te d in
im m unoprecipitation ex p erim ents on spent m edia.
A ltern ately , serum AGP could be adsorbed to ep ith elial cells in th e
p ro sta te , finding its w ay to the epithelium via the in te rs titia l spaces in vivo.
This would explain th e lack of d e tec ta b le bio sy n th etic p ro d u ct in vitro and the
positive im m unohistochem ical staining re a ctio n in tissue sectio n . The source
of AGP in tissue sectio n s m ay also be th e serum th a t spills from blood vessels
cut during p ro sta te resectio n . Highly glycosylated serum AGP could non-
sp ecifically adhere to ep ith elial cells through ionic or o th er in teractio n s.
T here is evidence th a t serum p ro tein s stick to cells in v itro (Irie, Irie and
M orton, 1974). H ow ever, sim ple adherence to cells does not explain th e
p atch y staining p a tte rn of positively stain ed cells in tissue sectio n s containing
136
p ro sta te carcinom a. P resen tly , the source of AGP antig en in p ro sta te sections
is unclear.
An analysis of the ex act isotype d istribution from b io sy n th etic sources
has not been perform ed. AGP, new ly synthesized by th e liver, m ay contain
m ore neuram inic acid residues than the m olecules which are already in blood
circu latio n . This m ay re la te to th e h a lf-life of AGP in blood since previous
w ork has shown th a t glycoproteins w ith term in al sialic acids rem oved are
quickly cleared from blood by liver parenchym al cells (Ashwell and M orell,
1971). The re le ase of new AGP into serum would th e re fo re explain the
app earan ce of an acidic sh ift. Since liver produces large am ounts of a cu te
phase p roteins, including AGP, during in flam m ato ry processes (Koj, 1974;
K indm ark, 1976), th e presence of an acidic sh ift m ay in d icate activ e in
flam m atio n or ongoing tissue d istru ctio n . The e x ten t of disease involvem ent
m ay influence th e size of th e acidic sh ift since advanced CAP p a tie n ts showed
la rg e r isotype ra tio s than did those w ith e arlier can cer stages.
The e x ac t biological function of AGP is n o t y e t established. H ow ever, it
m ay have a role in the im m une response, as AGP has been d em o n strated to
inhibit lym phoblasto gene sis and to gen erally suppress th e im m une system in an
anim al m odel (Chiu e t al., 1977; B en n ett and Schm id, 1980; Shibata, T am ura
and Ishida, 1983). The observations m ade in th is c h ap te r raise th e question of
w hether all AGP iso types display equal im m unosuppressive activ ity .
In sum m ary, AGP has been resolved into a t le a st 9 isotypes whose
d istribution changes in p a tie n t sera as com pared to norm al sera. A dditional
acidic isotypes can also be found in som e p a tie n t se ra b u t not in se ra from
h ealth y persons. T here appears to be no c o rrelatio n betw een th e observed
acidic sh ift, app earan ce of isotype 0 and th e to ta l co n cen tratio n of AGP. The
137
isotype d istrib u tio n p a tte rn m ay u ltim a te ly rev eal new in fo rm atio n about
in flam m ato ry or n eo p lastic disease w hich will supplem ent th e m onitoring of
AGP levels in serum .
138
CHAPTER VI
CONCLUSION
139
The p resen t study was u n d ertak en to broaden th e knowledge of p ro sta te
asso ciated p ro tein s and glycoproteins. A b e tte r understanding of th e norm al
and abnorm al functions of this gland is im p o rtan t since th e m ost serious
disease of th e p ro sta te , carcinom a, is th e second m ost com m on cause of death
by m alignancy in m ales in th e U nited S ta te s (Silverberg, 1982). Any
in v estig atio n th a t could lead to b e tte r diagnosis and tre a tm e n t of th is disease
is w orth pursuing.
This d issertatio n is a study w hich in v estig ated th e range of p ro sta te
asso ciated m olecules th a t have p o te n tia l use as p ro sta te organ or p ro sta te
disease m arkers. Such a search is pro m p ted by th e hypothesis th a t the
breakdow n of glandular tissue in te g rity leads to th e release of m acrom olecules
into surrounding tissues and blood, and th a t d etectio n of such com ponents can
be useful in diagnosis of adenocarcinom a and m onitoring of disease tre a tm e n t.
This inv estig atio n searched for p ro sta te asso ciated m olecules in four d ifferen t
sources, th e p ro sta tic fluid, th e spent m edia of p ro sta tic cell and organ
cu ltu res, and the serum .
A. PROSTATIC FLUID.
Since h y p erplastic p ro sta te m aintains nearly norm al th re e dim ensional
glandular stru c tu re , contains in ta c t basem ent m em brane and does not contain
m alignant cells, h y p erp lastic tissue and th e fluid co llected from it can be
considered n early norm al and suitable for description of p ro sta tic products.
H ow ever, since p ro sta tic fluid was co llected from re se c te d tissue, th e serum
p ro tein s w hich in ev itab ly mix w ith th e glandular fluid had to be id en tified .
Two dim ensional gel electro p h o resis, W estern b lo ttin g and im m unoautoradio-
graphic d e tec tio n of serum p ro te in antigens id en tified four of th e seven m ajor
140
p ro tein com ponents in p ro sta tic fluid as serum p ro tein s. T hree oth er m ajor
p ro sta tic fluid com ponents, resolved in th e pH range 5 to 7, do n o t have
antigenic, m olecular w eight or pi c h a ra c te ristic s in com m on w ith serum
p roteins. C onsidering th e source of th ese com ponents and th e sec re to ry
function of the p ro s ta te , th e conclusion is th a t th ese th re e non-serum p ro tein s
are p ro sta te asso ciated .
B. CREATINE KINASE - BB.
P ro sta tic fluid, known to contain larg e am ounts of c re a tin e kinase-BB
a ctiv ity , was shown in this study to be a suitable source for p u rificatio n of this
enzym e. The lo catio n of this com ponent on a 2D gel p a tte rn of p ro sta tic fluid
was also defined, and com pared to th e seven m ajor com ponents visualized on
gels, CK-BB was noted to be a relativ ely m inor p ro tein in the fluid. One
draw back of using p ro s ta tic fluid as a source of this isozym e is the contam ina
tion of the CK-BB p re p a ratio n w ith serum AGP, w hich m ixes w ith the fluid as
it is co llected from re se c te d p ro sta te tissue. A problem w ith p o te n tia l use of
CK-BB as p ro s ta te organ m ark er is th a t it is cro ssreactiv e w ith CK-BB
iso lated from brain. The p ro sta tic enzym e m ay prove useful if highly specific
antibodies can be found w hich can recognize antigenic d eterm in an ts ch arac
te ris tic of p ro sta tic CK-BB alone. This m ay be possible since another
p ro sta tic enzym e, p ro sta tic acid phosphatase, w as found to be im m unologically
d ifferen t from serum acid phosphatase (Schulman e t al., 1964).
141
C. BIOSYNTHETIC GLYCOPROTEIN PRODUCTS OF PROSTATE CELL
AND ORGAN CULTURES.
F resh hy p erp lastic p ro sta te tissue grow n in cu ltu re released sev eral
groups of m etab o lic ally radiolabeled glycoproteins. P ro sta tic carcin o m a cell
line PC-3 also in co rp o rated m etabolic precursors into glycoproteins which
w ere released into th e m edium . None of th e bio sy n th etic products w ere
im m unoprecipitable by antibodies to serum p ro tein s. Five of the com ponents
released in each system shared com m on m olecular w eight and pi ranges w hich,
when considering th e tissue source, in d icates th a t they m ay be id en tical
com ponents. In terestin g ly , th re e of th e ap p aren tly com m on glycoproteins also
shared th ese c h a ra c te ristic s w ith non-serum com ponents found in p ro sta tic
fluid. This fu rth e r suggests th a t th e glycoproteins released by cu ltu res in v itro
m ay be se c re to ry p ro ducts in vivo. Any of th ese th re e com ponents should be
considered prim e candidates for p ro sta te organ or disease m arkers and should
be studied fu rth er.
D. a -l-A C ID GLYCOPROTEIN ISOTYPES.
The d istrib u tio n of AGP p ro tein in th e se ra of p a tie n ts w ith p ro sta tic
diseases was found to sh ift significantly tow ards th e acidic isotypes. Also, a t
le a st one e x tra acidic isotype was seen in som e p a tie n t sera th a t was n o t
d e te c ta b le in norm al individuals. H ow ever, th e AGP acidic sh ift and the
app earan ce of an e x tra acidic isotype was also no ted in b re a st can cer p a tie n t
se ra and in th e sera of individuals w ith a chronic in flam m ato ry condition.
T h erefo re, th is sy stem ic change is not sp ecific for carcinom a. R a th e r it
appears re la te d to the re le ase of new ly sy n thesized m olecules into th e blood
system in p a tie n ts w ith d eg en erativ e conditions.
142
A sim ilar acidic sh ift was noted in th e p ro sta te asso ciated m olecule,
p ro sta tic acid phosphatase. By iso e le c tric focusing, m ore of th e re la tiv e ly
acidic isozym es w ere n o ted in both the serum and th e p ro sta tic tissue of
p a tie n ts w ith m alignant disease as com pared to norm al. Also, it w as seen th a t
sera having th e g re a te s t in crease in enzym e a ctiv ity also had th e la rg e st sh ift
tow ards acidic isozym es (Chu e t al., 1977; F o ti, H erschm an and C ooper, 1977;
Chu e t al., 1978). The in crease in p ro sta tic acid phosphatase a c tiv ity could
com e from new ly synthesized or released enzym e. As w ith AGP, th e freshly
produced m olecules m ay contain e x tra sialic acid m o ieties w hich cause th e
acidic sh ift. T h erefo re, it is possible th a t an acidic sh ift is a g en eral
c h a ra c te ris tic of new ly synthesized glycoproteins.
D eterm ining th e distrib u tio n of AGP isotypes m ay y et prove useful in
m onitoring th e course of can cers, as th e re was som e in d icatio n th a t the ex ten t
of th e disease c o rre la te s w ith th e m agnitude of th e acidic sh ift in advanced,
m e ta s ta tic carcinom a. F u rth e r work w ill d eterm in e if such a co rrelatio n can
be established and if it can be helpful in determ ining response to tre a tm e n t.
E. FINAL COMMENTS.
To d a te, no can cer-sp ecific p ro tein s have been found e ith e r fo r car
cinom a of th e p ro sta te or for carcinom a of any o th er organ. H ow ever,
num erous tu m o r-asso ciated antigens, w hich are norm al organ products, have
been re p o rte d . M onitoring th e level of th ese antigens in th e sera of can cer
p a tie n ts has proved valuable in follow ing disease rem ission or relapse in
p a tie n ts undergoing chem otherapy (Gropp e t al., 1977). F or th e p ro sta te ,
p ro s ta tic acid phosphatase and p ro sta te antigen have proven m ost useful
(Choe e t al., 1982; Wang e t al., 1982). T hat both of th ese antigens are found
143
to be elev ated in sera of p ro s ta te carcinom a p a tie n ts supports the hypothesis
th a t breakdow n of tissue in te g rity allows leakage of organ products into blood
circulation. The d e tec tio n of th ese tw o antigens has also been no ted in tissue
sections (Choe e t al., 1982; Wang et al., 1982), in d icatin g th e p o te n tia l u tility
of p ro sta tic products in h istochem ical id en tificatio n of th e prim ary sites of
d istan t m e ta sta tic gen ito u rin ary tum ors.
H ow ever, no sim ple serum te st is y e t available to d e te c t th e e arliest,
m ost curable stag es of p ro sta te carcinom a, or to id en tify individuals w ith high
risk of disease. Such a te s t m ay be im possible to develop for the following
th e o re tic a l reasons. F irst, a sm all w e ll-d ifferen tia te d carcin o m a m ay disrupt
an insu fficien t a re a w ithin a tissue to allow organ-or can cer-asso ciated
m olecules to escape from th e can cer site. Second, a sm all focus of carcinom a
cells m ay not release enough antigenic m a te ria l into th e surrounding stro m a to
be d e te c te d , even w hen th e organ stru c tu re is adeq u ately destroyed. Such a
sm all carcinom a m ay re le ase inadequate am ounts of m a te ria l so th a t the
antigens rem ain u n d e tec ta b le even by th e m ost sen sitiv e serum im m unoassays.
Third, a larg e hyp erp lastic grow th m ay cause enough tissue com pression and
physical dam age to allow norm ally seq u estered organ p ro ducts to leak into th e
surrounding stro m a. This o ccu rrence would destroy th e sp ec ific ity of a cancer
te s t based on leakage of tissue com ponents into th e blood system .
From th ese considerations, even th e m ost sensitive and specific serum
assays for can cer m ay be unable to d e te c t early disease stag es in su fficien t
num bers of p a tie n ts. R ecen t studies on p ro sta tic acid phosphatase support this
conclusion (H erschm an, 1980). F or exam ple, only 24% of p a tie n ts w ith early
p ro sta te carcinom a showed elev ated serum levels of this enzym e (G riffiths,
1982).
144
H ow ever, th e general search fo r organ- or c an cer-asso ciated products
should still be continued for th e follow ing reasons. T here is still th e possibility
th a t a tru ly can cer-sp ecific antigen m ay be found. The d escription of new
o rg an -asso ciated antigens w ill be useful in im m unohistochem ical id e n tific a tio n
of a m e ta s ta tic tum or's p rim ary s ite . M onitoring th e serum levels of m any
c an cer-asso ciated p ro tein s has, as m entioned above, been u tilized in d e te c tin g
disease relap se. As w ell, a num ber of te sts fo r organ- and can cer-asso ciated
m olecules could be com bined in a te s t panel w hich could im prove th e
frequency of carcin o m a d etectio n . Also, highly sp ecific im m unoglobulins, such
as m onoclonal antibodies, w hich recognize can c e r-a sso c iated antigens could be
used fo r specific im m unotherapy. Studies in any of th ese various areas m ay
even tu ally lead to im proved tre a tm e n ts and in creased num bers of cures in
can cer p a tie n ts.
145
REFERENCES
A leyassine, H. and M ad ssa c , S.G. The diagnostic significance of serum
c reatin e kinase-BB isozym e in adenocarcinom a of th e p ro sta te . Clin.
Biochem . 13: 109-112, 1980.
A ltland, K ., R oeder, Th., Jakin, H.M., Zim m er, H.-G. and N euhoff, V. Demon
stra tio n of a- 1-acid glycoprotein (orosomucoid) by double one-dim ensional
slab gel electrophoresis: Evidence for in tra - and interindividual variab ility
of th e m icro h etero g en eity p a tte rn in h e alth and disease. C lin. C hem . 28:
1000- 1010, 1982.
A nderson, L. and A nderson, N.G. High resolution tw o-dim ensional electropho
resis of hum an plasm a p roteins. P roc. N atl. A cad. Sci. USA 74: 5421-5425,
1977.
Anderson, N.L. and A nderson, N.G. M icro h etero g en eity of serum tran sferrin ,
haptoglobin and alpha-2-H S glycoprotein exam ined by high resolution tw o
dim ensional gel electro p h o resis. Biochem. Biophys. R es. Com m un. 88: 258-
265, 1979.
A nderson, R.U. and F a ir, W .R. Physical and chem ical determ in atio n of
p ro sta tic secretio n s in benign hyperplasia, p ro sta titis and adenocarcinom a.
Invest. U rol. 14: 137-140, 1976.
146
A rm strong, J.B ., Lowden, J.A . and Sherwin, A.L. B rain isozym e of c re a tin e
kinase. I. P u rificatio n o f rab b it enzym e and production of specific an ti
bodies. J. Biol. Chem . 252: 3105-3111, 1977.
Ashwell, G. and M orell, A.G. G alactose: A cry p tic d eterm in an t of glycopro
tein catabolism . In: G.A. Jam ieson and T .J. G reenw alt (eds.), G lycoproteins
of Blood C ells and Plasm a, pp. 173-189. Philadelphia: L ippincott, 1971.
A tanasov, N.A. and Gikov, D.G. D isc electro p h o resis of p ro sta tic fluid.
R elativ e m obility and m olecular w eight of som e enzym es and p ro tein s. Clin.
Chim . A cta 36: 213-221, 1972.
A um uller, G. and Adler, G. E xperim ental studies o f apocrine secretio n in th e
dorsal p ro sta te epithelium o f th e ra t. C ell Tissue Res. 198: 145-148, 1979.
B alerna, M., Colpi, G.M., C am pana, A., R oveda, L., Tom m asini-D egna, A. and
Zanollo, A. H igh-resolution p ro te in p a tte rn s o f hum an expressed p ro sta tic
secretio n : A new to o l for the diagnosis of p ro sta titis. Arch. Androl. 8: 97-
105, 1982.
Ban, Y., Wang, M.C. and Chu, T.M. B iological n a tu re of th e hum an p ro s ta te -
specific antigen. Fed. Proc. 42: 406, 1983, A bstr. 669.
B ennett, M. and Schmid, K. Im m unosuppression by hum an plasm a alpha-1-acid
glycoprotein: Im portance o f the carbohydrate m oiety. Proc. N atl. Acad. Sci.
USA 77: 6109-6113, 1980.
B radford, M. A rapid and sensitive m ethod for th e q u an tita tio n of m icrogram
q u an titie s o f p ro tein u tilizin g th e principle o f p ro tein -d y e binding. Anal.
Biochem . 72: 248-254, 1976.
Brandes, D. and K ircheim , D. H istochem istry of th e p ro sta te . In: M.
Tanneribaum (ed.), U rologic Pathology: The P ro sta te , pp. 99-128. P hiladel
phia: Lea and Febiger, 1977.
B u rn ette, W.N. "W estern B lotting": E lectro p h o retic tra n sfe r o f p ro tein s from
sodium dodecyl sulfate-p o ly acry lam id e gels to unm odified n itro cellu lo se and
radiographic d e tec tio n w ith antibody and radioiodinated p ro te in A. Anal.
Biochem . 112: 195-203, 1981.
C a rte r, D.B. and Resnick, M.I. High reso lu tio n analysis of hum an p ro sta tic
fluid by tw o-dim ensional electro p h o resis. P ro sta te _ 3 : 27-33, 1982.
Chiu, K.M., M ortensen, R .F ., Osm and, A .P. and G ewurz, H. In te ra c tio n s of
alpha-1-acid glycoprotein w ith the im m une system . I. P u rificatio n and
e ffe c ts upon lym phocyte responsiveness. Im m unology 32: 997-1005, 1977.
Choe, B.K., Pontes, E .J., M cDonald, L and R ose, N.R. P u rificatio n and
c h a ra c te riz a tio n of hum an p ro sta tic acid phosphatase. Prep. Biochem . 8: 73-
89, 1978.
Choe, B.K., Pontes, E .J., Lillehoj, H.S. and Rose, N.R. Im m unohistological
approaches to hum an p ro s ta tic ep ith elial cells. P ro sta te D 383-398, 1980.
148
C hoe, B.K. and R ose, N.R. P ro sta tic acid phosphatase: A m arker for hum an
p ro sta tic adenocarcinom a. M eth. C ancer Res. 1^: 199-231, 1982.
Chu, T.M ., Wang, M .C., K uciel, R ., V alenzuela, L. and Murphy, G.P. Enzym e
m arkers in hum an p ro sta tic carcinom a. C ancer T reat. Rep. 6R 193-200,
1977.
Chu, T.M., Wang, M .C., M errin, C., V alenzuela, L. and Murphy, G.P. Isozym es
o f hum an p ro sta tic acid phosphatase. Oncology 35: 198-200, 1978.
Coolen, R.B. P ragay, D.A., N osanchuk, J.S. and Belding, R. E levation of
b ra in - type c re a tin e kinase in serum from p a tie n ts w ith carcinom a. C ancer
44: 1414- 1418, 1979.
C ooper, E.H. and Stone, J. A cute phase re a c ta n t p ro te in s in cancer. Adv.
C ancer Res. 30: 1-44, 1979.
Cullen, S.E. and Schw artz, B.D. An im proved m ethod for isolation of H-2 and
la alloantigens w ith im m unoprecipitation induced by p ro tein A -bearing
staphylococci. J. Im m unol. 117: 136-142, 1976.
D enis, L. J ., P rout, G.P. Jr., Van Camp, K. and Van Sande, M. E lectro p h o retic
c h a ra c te riz a tio n o f p ro s ta te : P ro tein and la ctic dehydrogenase in benign
hyperplasia and carcinom a. J. Urol. 88: 77-85, 1962.
149
D erm er, G.B., Silverm an, L.M. and Chapm an, J.F . E nhancem ent techniques
for d etectin g tra c e and fluid-specific com ponents in tw o-dim ensional e lec
tro p h o retic p a tte rn s. Clin. Chem . 28: 759-765, 1982.
D ubray, G. and B ezard, G. A highly sensitive periodic acid -silv er stain for 1,2-
diol groups o f glycoproteins and polysaccharides in polyacrylam ide gels.
Anal. Biochem . 119s 325-329, 1982.
Elder, J.H ., P ic k e tt, R .A ., H am pton, J. and L em er, R.A. R adioiodination of
p ro tein s in single polyacrylam ide gel slices. T ryptic p ep tid e analysis o f all
th e m ajor m em bers of com plex m ulticom ponent system s using m icrogram
q u a n titie s o f to ta l p ro tein . J. Biol. Chem . 252: 6510-6515, 1977a.
Elder, J.H ., Jensen, F .C ., B ryant, M.L. and L em er, R.A. Polym orphism o f th e
m ajor envelope glycoprotein (gp 70) of m urine C -type viruses: virion associ
ated and d ifferen tia tio n antigens encoded by a m ulti-gene fam ily. N ature
267: 23-28, 1977b.
E levitch, F .R . F lu o rom etric techniques in clinical chem istry. Boston: L ittle,
Brown and Co., 1973.
Eliasson, R. A ccessory glands and sem inal plasm a w ith special re fe re n c e to
in fe rtility as a m odel for studies on induction of ste rility in th e m ale. J.
Reprod. F e rtil. (SuppL) 24: 163-174, 1976.
150
Fairbanks, G., Steck, T.L. and W allach, D .F.H. E lectro p h o retic analysis o f th e
m ajor polypeptides of the hum an ery th ro c y te m em brane. B iochem istry 10:
2606-2617, 1971.
Feld, R.D ., Van S teirteghem , A .C., Zweig, M.H., W eim ar, G.W., N arayana,
A.S. and W itte, D.L. The presen ce of c re a tin e kinase BB isozym e in p a tie n ts
w ith p ro sta tic cancer. Clin. Chim. A cta 100: 267-273, 1980.
Feld, R.D. and W itte, D.L. P resence o f c re a tin e kinase BB isozym e in some
p a tie n ts w ith p ro sta tic carcinom a. Clin. Chem. 23: 1930-1932, 1977.
Form an, D.T. The significance of c re a tin e kinase (CKBB) in m e ta sta tic cancer
of the p ro sta te . Ann. Clin. Lab. Sci. 9: 333-337, 1979.
Foti, A.G., H erschm an, H. and Cooper, J.F . Isozym es o f acid phosphatase in
norm al and cancerous hum an p ro sta tic tissue. C ancer Res. 37: 4120-4124,
1977.
F o u m et, B., M ontreuil, J., S treck er, G., Dor land, L., H averkam p, J., V liegent-
h a rt, J.F .G ., B in ette, J.P . and Schmid, K. D eterm in atio n of the prim ary
stru c tu re s of 16 asialo-carbohydrate units derived from hum an plasm a a -1 -
acid glycoprotein by 360-MHz NMR spectroscopy and p erm eth y latio n
analysis. B iochem istry 17: 5206-5214, 1978.
Frank, R.N. and Rodbard, D. Precision of sodium dodecyl su lfate-p o ly acry l-
am ide-gel electrophoresis for the m olecular w eight estim atio n of a m em
151
brane glycoprotein: Studies on bovine rhodopsin. Arch. Biochem . Biophys.
*
171: 1-13, 1975.
Franks, L.M. Etiology and epidem iology of hum an p ro sta tic disorders, hi: M.
Taim enbaum (ed.), U rologic Pathology: The P ro sta te , pp. 23-29. P hiladel
phia: Lea and Febiger, 1977.
Franks, L.M. P rim ary cu ltu res of hum an p ro sta te . M eth. C ell Biol. 21B: 153-
169, 1980.
G ahm berg, C.G. and A nderson, L.C. L eukocyte surface origin of hum an a -1 -
acid glycoprotein (orosom ucoid). J. Exp. Med. 148: 507-521, 1978.
Gordon, A.H. and Dykes, P .J. a -1 -A c u te phase globulins of ra ts. M icrohetero
gen eity a fte r iso elec tric focusing. Biochem . J. 103: 95-101, 1972.
G ranger, B.L. and L azarides, E. D esm in and vim entin coexist a t the p reiphery
o f the m yofibril Z disc. C ell 18: 1053-1063, 1979.
G rayhack, J.T . and Lee, C. E valuation of p ro sta tic fluid in p ro sta tic
pathology. In: G.P. M urphy, A.A. Sandberg, J.P . K arr (eds.), The P ro sta tic
C ell: S tru ctu re and Function, P a rt A, pp. 231-246. New York: Alan R. Liss,
Inc., 1981.
G rayhack, J.T ., Lee, C., Kolbusz, W. and O liver, L. D etectio n of carcinom a of
th e p ro sta te using biochem ical observations. C ancer 45: 1896-1901, 1980.
152
G rayhack, J.T ., Wendel, E.F., Lee, C. and O liver, L. Analysis of p ro sta tic
fluid in p ro sta tic disease. C ancer T reat. Rep. 61: 205-210, 1977a.
G rayhack, J.T ., W endel, E.F., Lee, C., O liver, L. and Cohen, E. L a c ta te
dehydrogenase isoenzym es in hum an p ro sta tic fluid: An aid in recognition of
m alignancy? J. UroL 118: 204-208, 1977b.
G rayhack, J.T ., W endel, E .F., O liver, L. and Lee, C. Analysis of specific
p ro tein s in p ro sta tic fluid for d e tec tin g p ro sta tic m alignancy. J. Urol. 121:
295-299, 1979.
G riffith s, J. The ap p ro p riate uses of p ro sta tic acid phosphatase d eterm in a
tions in the diagnosis o f adenocarcinom a of the p ro sta te . Ann. N.Y. Acad.
Sci. 390: 100-103, 1982.
Gropp, C., Lehm ann, F.G ., B auer, H.W. and Havem ann, K. C arcinoem bryonic
antigen, a -1 -fe to p ro te in , fe rritin and a-2 -p reg n an cy asso ciated glycoprotein
in th e serum o f lung cancer p a tie n ts and its d em onstration in lung tum or
tissue. Oncology 34: 267-272, 1977.
G utm an, E.B., Sproul, E.E. and G utm an, A.B. Increased phosphatase activ ity
o f bone a t site o f o steo p lastic m e ta sta ses secondary to carcinom a o f p ro sta te
gland. Am. J. C ancer 28: 485-495, 1936.
153
G utm an, A.B. and G utm an, E.B. An "acid" phosphatase occurring in the serum
o f p a tie n ts w ith m etastasizin g carcinom a of th e p ro sta te gland. J. Clin.
Invest. 17: 473-478, 1938.
H ager, D.A. and Burgess, R .R . E lution of p ro te in s from sodium dodecyl
su lfate-polyacrylam ide gels, rem oval of sodium dodecyl su lfate and
re n a tu ra tio n of E scherichia coli RNA polym erase, w heat germ DNA
topoisom erase, and o th er enzym es. Anal. Biochem . 109: 76-86, 1980.
H eat field, B.M., Sanefuji, H. and Trump, B.F. L ong-term explant cu ltu re of
norm al hum an p ro sta te . M eth. C ell Biol. 2 IB : 171-194, 1980.
H erschm an, H .R. P ro sta tic acid phosphatase and p ro sta tic disease. T rends
Biol. Sci. _ 5 : 82-84, 1980.
H om burger, H.A., M iller, S.A. and Jacob, G.L. R adioim m unoassay of c re atin e
kinase B -isozym es in serum o f p a tie n ts w ith azotem ia, o b structive uropathy
or carcinom a of th e p ro sta te or bladder. Clin. Chem . 26: 1821-1824, 1980.
Hudson, P.B ., Finkle, A.L., Hopkins, J.A ., Sproul, E.E. and Stout, A.P.
Pro sta tic cancer. XL Early p ro sta tic can cer diagnosed by a rb itra ry open
p e rin e al biopsy am ong 300 u n selected p a tie n ts. C ancer Ji 690-703, 1954.
H unter, W.M. and Greenwood, F.C . P rep a ra tio n of iodine-131 labeled hum an
grow th horm one o f high specific activ ity . N ature 194: 495-496, 1962.
154
Im am , A., L aurence, D .J.R . and N eville, A.M. Isolation and c h a ra c te riz a tio n
o f a m ajor glycoprotein from m ilk -fat-globule m em brane o f hum an b re a st
m ilk. Biochem . J. 193; 47-54, 1981.
Irie, R .F ., Irie, K. and M orton, D.L. N atu ral antibody in hum an serum to a
neoantigen in hum an cu ltured cells grown in fe ta l bovine serum . J. N atl.
C ancer Inst. 52: 1051-1058, 1974.
Jam ieson, J.C . and Ashton, F.E. Studies on a cu te phase p ro tein s o f r a t serum ,
m . Site of synthesis of album in and a -1 -a c id glycoprotein and the co n ten ts of
th ese p ro tein s in liver m icrosom e fractio n s from ra ts suffering from induced
inflam m ation. Can. J. Biochem . 51: 1034-1045, 1973a.
Jam ieson, J.C . and Ashton, F.E . Studies on acu te phase p ro tein s o f r a t serum .
IV. P athw ay of secretio n o f album in and a -1 -a c id glycoprotein from liver.
Can. J. Biochem . 51: 1281-1291, 1973b.
Kaighn, M.E., Shanker N arayan, K., Ohnuki, Y., Lechner, J.F . and Jones, L.W.
E stablishm ent and c h a ra c te riz a tio n of a hum an p ro sta tic carcinom a cell line
(PC-3). Invest. U rol. 17: 16-23, 1979.
K am iyam a, S. and Schmid, K. Studies on th e stru c tu re of a -1 -a c id glycopro
tein. IE. P rep aratio n and c h a ra c te riz a tio n o f a glycopeptide fractio n .
Biochim . Biophys. A cta 58: 80-87, 1962a.
155
K am iyam a, S. and Schmid, K. Studies on th e stru c tu re of a -1 -a cid glycopro
tein. V. The linkage betw een th e polypeptide and carbohydrate m oiety.
Biochim. Biophys. A cta 63: 266-275, 1962b.
K ent, J.R ., Hill, M., B ischoff, A. Acid phosphatase co n ten t of p ro sta te
expirm ate from p a tie n ts w ith advanced p ro sta tic carcinom a: A p o te n tia l
prognostic th erap eu tic index. C ancer 25: 858-862, 1970.
K essler, S.W. R apid isolation of antigens from cells w ith a staphylococcal
p ro tein A -antibody adsorbant: P a ra m e te rs of the in teractio n of antibody-
antigen com plexes w ith p ro tein A. J. Imm unol. 115: 1617-1624, 1975.
K essler, S.W. C ell m em brane antigen isolation w ith the staphylococcal p ro tein
A -antibody adsorbant. J. Im m unol 117: 1482-1490, 1976.
K halil, A., Wilson, M .J., Goneli, S.A. and S teer, R .C . Functional B iochem istry.
In: E. Spring-M ills, E.S.E. H afez (eds.), A ccessory Glands of th e Male
R eproduction T ract, pp. 69-108, Ann Arbor: Ann Arbor Science Publishers,
1979.
K im ler, S.C. and Sandhu, R.S. C irculating CK-BB and CK-MB isozym es a fte r
p ro sta te resectio n . Clin. Chem . 26: 55-59, 1980.
Kindm ark, C.O. Sequential changes in plasm a p ro tein s in various acute
diseases. In: R. Bianchi, G. M ariani and A.S. M cFarlane (eds.), Plasm a
P ro tein Turnover, pp. 395-402, B altim ore: U niversity P ark Press, 1976.
156
Koj, A. A cute phase re a c ta n ts. In: A.C. Allison (ed.), S tru ctu re and Function
o f Plasm a P roteins, Vol. 1, pp. 93-95. New York: Plenum Publishing C orp.,
1974.
K ostka, V. and C arp en ter, F.H . Inhibition of chym otryptic a ctiv ity in c ry sta l
line trypsin p rep aratio n s. J. Biol. Chem . 239: 1799-1803, 1964.
Krohn, K.A., Knight, L.C ., Harwig, J.F . and Welch, M .J. D ifferen ces in the
sites of iodination of p ro tein s following four m ethods of radioiodination.
Biochim. Biophys. A cta 490: 497-505, 1977.
K ronvall, G., Seal, U.S., F instad, J. and W illiams, R .C . Jr. P hylogenetic
insight into evolution of m am m alian Fc fragm ent of gam m a G globulin using
staphylococcal p ro tein A. J. Im m unol. 104: 140-147, 1970.
K uriyam a, M., Wang, M.C., Papsidero, L.D ., K illian, C.S., Shimano, T., Valen
zuela, L., N ishiura, T., Murphy, G.P. and Chu, T.M. Q u an titatio n of p ro s ta te -
specific antigen in serum by a sensitive enzym e im m unoassay. C ancer Res.
40: 4658-4662, 1980.
L ascelles, A.K. Role of th e m am m ary gland and milk in immunology. In: M.
P eak er (ed.), C om parative A spects of L actatio n , pp. 241-260. New York:
A cadem ic Press, Inc., 1977.
L aurence, D .J.R . Milk p ro tein s and m am m ary can cer: A review . Invest. Cell
P ath o l., 1 .: 5-22, 1978.
157
Moss, B.A. P ep tid e m apping a t th e nanom ole level. In: I. L efkovits and B.
P ern is (eds.), Im m unological M ethods, pp. 69-80. New York, A cadem ic Press,
1979.
N aritoku, W. Im m unohistologic lo calization of im m unoglobulins G, A and M,
J-chain and sec re to ry com ponent in th e epithelium of benign and m alignant
hum an p ro sta tes. In: Im m unoperoxidase Study of P ro sta te C ancer, pp. 37-
50. Los Angeles: U niversity of Southern C alifornia, 1982.
N aritoku, W.Y. and Taylor, C .R. A co m p arativ e study of the use of
m onoclonal antibodies using th re e d iffe re n t im m unohistochem ical m ethods:
An evaluation of m onoclonal and polyclonal antibodies against hum an pros
ta tic acid phosphatase. J. H istochem . C ytochem . 30: 253-260, 1982.
N eri, A., R uoslahti, E. and Nicolson, G.L. D istribution of fib ro n ectin on clonal
cell lines of a ra t m am m ary adenocarcinom a grow ing in v itro and in vivo a t
p rim ary and m e ta sta tic sites. C ancer Res. 41: 5082-5092, 1981.
N icollet, I., L ebreton, J.-P ., Fontaine, M. and Hiron, M. Evidence for alpha-1-
acid glycoprotein populations of d iffe re n t p i values a fte r C oncanavalin A
affin ity chrom atography. Study of th eir evolution during inflam m ation in
man. Biochim. Biophys. A cta 668: 235-245, 1981.
N ylander, G. The e lec tro p h o retic p a tte rn of p ro sta tic p ro tein s in norm al and
pathologic secretio n . A cta Chir. Scand. 109: 473-482, 1955.
158
O 'F arrell, P.H . High resolution tw o-dim ensional electrophoresis of p roteins.
J. Biol. Chem . 250: 4007-4021, 1975.
O strow ski, W., Wasyl, Z., W eber, M., Guminska, M. and L uchter, E. The role
o f neuram inic acid in th e h e tero g en e ity o f acid phosphom onoesterase from
th e hum an p ro sta te gland. Biochim . Biophys. A cta 221: 297-306, 1970.
Papsidero, L.D., K uriyam a, M., Wang, M .C., H oroszew icz, J., Leong, S.S.,
V alenzuela, L., Murphy, G.P. and Chu, T.M. P ro sta te antigen: A m ark er for
hum an p ro s ta te ep ith elial cells. J. N atl. C ancer Inst. 66: 37-42, 1981.
P arry, W.L. P ro sta te m alignancies. In: J.F . Glenn (ed.), Urologic Surgery, pp.
546-588. H agerstow n: H arper and Row, 1975.
P retlow , T.G., W hilehurst, G.B., P retlow , T.P., Stillw ell Hunt, R ., Jacobs,
J.M ., M cKenzie, D .R., M cDaniel, H.G., Hall, L.M. and B radley, E.L. Jr.
D ecrease in c re atin e kinase in hum an p ro sta tic carcinom a com pared to
benign p ro sta tic hyperplasia. C ancer R es. 42: 4842-4848, 1982.
P rout, G.R. Jr. Diagnosing and staging o f p ro sta tic carcinom a, hi: M.
Tanenbaum (ed.), U rologic Pathology: The P ro sta te , pp. 199-213. Phila
delphia: Lee and Febiger, 1977.
Putnam , F.W. P ersp ectiv es - p a st, p re sen t and fu tu re, hi: F.W. P utnam (ed.),
The P lasm a P roteins. S tru ctu re, F unction and G enetic C ontrol, Vol. 1, pp. 1-
55. New York: A cadem ic Press, 1975.
159
R esnick, M.I. and Stubbs, A .J. Age specific e lectro p h o retic p a tte rn s o f
p ro sta tic fluid. J. Surg. R es. 24: 415-420, 1978.
Reynolds, J.A . and Tanford, C. Binding of dodecyl su lfate to p ro te in s a t high
binding ratio s. Possible im plications for the s ta te o f p ro tein s in biological
m em branes. P roc. N atl. A cad. Sci. USA 66: 1002-1007, 1970.
R ig h etti, P.G. and C aravaggio, T. Iso electric po in ts and m olecular w eights of
proteins. A tab le. J. C hrom atog. 127: 1-28, 1976.
Rosalki, S.B. An im proved procedure for serum c re a tin e phosphokinase
d eterm in atio n . J. Lab. Clin. Med. 69? 696-705, 1967.
Rudm an, D ., T readw ell, P.E ., Vogler, W .R., Howard, C.H. and Hollins, B. An
abnorm al orosom ucoid in th e plasm a of p a tie n ts w ith neoplastic disease.
C ancer Res. 32: 1951-1959, 1972.
35
Sato, C.S. and Gyorkey, F. S-labeled glycosam inoglycans of th e mouse
p ro sta te and its secretio n by organ cu ltu re (40712). Proc. Soc. Exp. Biol.
Med. 1 j63: 1-9, 1980.
S chaffner, C .P. P ro sta tic ch o lestero l m etabolism : R egulation and a lte ratio n .
In: G.P. Murphy, A.A. Sandberg, J.P . K arr (eds.), The P ro sta tic Cell:
S tru ctu re and Function, P a rt A, pp. 279-324. New York: Alan R. Liss, Inc.,
1981.
160
Schmid, K., B in ette, J.P ., K am iyam a, S., P fiste r, V. and Takahashi, S. Studies
on the stru c tu re of a - l- a c id glycoprotein. DI. Polym orphism of a - l- a c id
g lycoprotein and the p a rtia l reso lu tio n and c h a ra c te riz a tio n of its varian ts.
B iochem istry Jh 959-966, 1962.
Schmid, K., K aufm an, H., Isem ura, S., Bauer, F., Em ura, J., M otoyam a, T.,
Ishiguro, M. and Nanno, S. S tru ctu re o f a - l- a c id glycoprotein. The com plete
am ino acid sequence, m ultiple am ino acid substitutions, and hom ology w ith
im m unoglobulins. B iochem istry 12: 2711-2724, 1973.
Schmid, K. a -l-A c id glycoprotein. In: F.W. Putnam (ed.), The P lasm a
P roteins, Vol. 1, pp. 183-228. New York: A cadem ic Press, 1975.
S cott, R. Jr., M utchnik, D.L., Laskowski, T.Z. and Schm alhorst, W.R. C ar
cinom a o f the p ro sta te in elderly men: Incidence, grow th c h a ra c te ristic s and
clinical significance. J. Urol. 101: 602-607, 1969.
Shibata, Y., Tam ura, K. and Ishida, N. In vivo analysis of the suppressive
e ffe c ts of im m unosuppressive acidic p ro tein , a type of a - l- a c id glycoprotein,
in connection w ith its high level in tum or-bearing m ice. C ancer Res. 4 3 :
2889-2896, 1983.
Shulman, S., M amrod, L., Gonder, M .J. and Soanes, W.A. The d e te c tio n of
p ro sta tic acid phosphatase by antibody reactio n s in gel diffusion. J.
Imm unol. 93^: 474-480, 1964.
161
Silverberg, E. C ancer S tatistics, 1982. CA-A C ancer J. Clin. 32: 15-32, 1982.
Silverm an, L.M., C aruso, L.M ., Irwin, L.E., K itzm an, M. and Pincus, F.E.
C reatin e kinase BB isozym e a c tiv ity in bone m arrow serum . Clin. Chem . 24:
1423-1425, 1978.
Silverm an, L.M., D erm er, G.B., Zweig, M.H., Van S teirteghem , A.C. and
Tokes, Z.A. C reatin e kinase BB: A new tu m o r-asso ciated m arker. Clin.
Chem . 25: 1432-1435, 1979.
Sm ith, A.F. S eparation of tissu e and serum c re atin e kinase isoenzym es on
polyacrylam ide gels. Clin. Chim . A cta 39: 351-359, 1972.
Soanes, W.A., Shulman, S., M amrod, L., Barnes, G.W. and Gonder, M .J.
E lectro p h o retic analysis o f p ro sta tic fluid. Invest. Urol. L 269-278, 1963-64.
Tam ura, K., Shibata, Y., M atsuda, Y. and Ishida, N. Isolation and c h a ra c te ri
zatio n o f an im m unosuppressive acid p ro tein from asc itic fluids o f can cer
p a tie n ts. C ancer Res. 41: 3244-3252, 1981.
Taylor, C .R. Im m unoperoxidase Techniques. A rch. P athol. Lab. Med. 102:
113-121, 1978.
T aylor-P apadim itriou, J., B urcell, J. and H urst, J. Production of fibronectin
by norm al and m alignant hum an m am m ary ep ith elial cells. C ancer Res. 41:
2491-2500, 1981.
162
Tokes, Z.A. and D erm er, G.B. G lycoprotein synthesis as a function of
ep ith elial cell arran g em en t: B iosynthesis and re le ase o f glycoproteins by
hum an b re a st and p ro sta te cells in organ cu lture. J. Supram ol. S tru ct. 7;
515-530, 1977.
Tokes, Z.A., G endler, S.J., Im am , A., Pullano, T.G. and Ross, K.L. D ecipher
ing can cer-asso ciated glycoproteins which may have diagnostic relev an ce. In
press: P. Moloy and G.L. N icolson (eds.), C ellular Oncology, New A pproaches
in Biology, Diagnosis and T reatm en t, New York: P raeg er Press, 1983.
Towbin, H., Staehelin, T. and Gordon, J. E lectro p h o retic tra n sfe r of p ro tein s
from polyacrylam ide gels to nitro cellu lo se sheets: P rocedure and some
applications. Proc. N atl. Acad. Sci. USA 76: 4350-4354, 1979.
Tsung, S.H. C reatin e kinase isozym e p a tte rn s in hum an tissue obtained a t
surgery. Clin. Chem. 22: 173-175, 1976.
Wang, M .C., V alenzuela, L.A., Murphy, G.P. and Chu, T.M. P u rificatio n o f a
hum an p ro sta te specific antigen. Invest. Urol. 17: 159-163, 1979.
Wang, M.C. K uriyam a, M., P apsidero, L.D., Moor, R.M ., V alenzuela, L.A.,
Murphy, G.P. and Chu, T.M. P ro sta te antigen of hum an can cer p a tie n ts.
M eth. C ancer R es. 19: 179-197, 1982.
Wray, W., Boulikas, T., Wray, V.P. and H ancock, R. Silver staining of p ro tein s
in polyacrylam ide gels. Anal. Biochem . 118: 197-203, 1981.
163
Zaneveld, L .J.D . and Tauber, P .F . C ontribution of p ro sta tic fluid com ponents
to th e e ja c u la te . In: G.P. M urphy, A.A. Sandberg, J.P . K arr (eds.), The
P ro sta tic C ell: S tru ctu re and Function, P a rt A, pp. 265-277. New York:
Alan R. Liss, Inc., 1981.
Zweig, M.H., Van S teirteghem , A.C. and S checter, A.N. R adioim m unoassay of
c re a tin e kinase isozym es in hum an serum : Isozym e BB. Clin. Chem . 24: 422-
428, 1978.
Zweig, M.H. and Van S teirteghem , A.C. A ssessm ent by radioim m unoassay of
serum c re a tin e kinase BB (CK-BB) as a tum or m arker: Studies in p a tie n ts
w ith various can cers and a com parison of CK-BB co n cen tratio n s to p ro sta tic
acid phosphatase co n cen tratio n s. J. N atl. C ancer Inst. 66: 859-862, 1981.
164
UMI Number: DP21614
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
Dissertation Publishing
UMI DP21614
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code
ProQuest:
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106-1346

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The mechanism of alpha-chymotrypsin acylation by N-acylimidazoles

PDF

The metabolism in vitro of 4-C¹⁴-cytidine

PDF

A new approach to the problem of measuring the properties of micelles

PDF

The isolation and characterization of rat plasma albumins

PDF

The intermediary metabolism of butyric acid as determined from the physiological behavior of the various four carbon acids

PDF

The effects of irradiation upon lipid and carbohydrate metabolism in the rat

PDF

Protein-protein interactions in blood serum

PDF

The genes for the glucose-regulated protein GRP94 and GRP78 are co-ordinately regulated by common trans-acting factors

PDF

The incorporation of radioactive carbons of glucose into the protein-bound amino acids of one-day-old mouse brain

PDF

¹⁹F-NMR studies of trifluoroacetyl insulin derivatives

PDF

Characterization of glycoproteins synthesized by human breast epithelial cells

PDF

Isolation and characterization of intestinal alkaline phosphatase

PDF

Computer modeling and free energy calculatons of biological processes

PDF

The comparative glycogenetic and ketolytic action of glucose and some carbohydrate intermediates

PDF

The role of the adrenal cortex upon fluid shifts through the capillary wall

PDF

A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

PDF

Isolation and characterization of mouse GRIP1, a novel transcriptional coactivator of steroid receptors

PDF

The comparative ketolytic action of glucose, galactose, and lactose when administered to rats suffering from an artificial ketosis

PDF

Studies on cholesterol metabolism in the rat

PDF

The metabolism of the two di-deuterobutyric acids as indicated by the deuterium content of the excreted betahydroxbutyric acid

Asset Metadata

Creator Pullano, Thaddeus George (author)

Core Title The biochemistry of glycoproteins from human prostate

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1983-10

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-4441

Unique identifier UC11348850

Identifier DP21614.pdf (filename),usctheses-c17-4441 (legacy record id)

Legacy Identifier DP21614.pdf

Dmrecord 4441

Document Type Dissertation

Rights Pullano, Thaddeus George

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA