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Studies on the pulmonary surface-active lipoprotein: isolation and characterization of its peptide moiety

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Studies on the pulmonary surface-active lipoprotein: isolation and characterization of its peptide moiety

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Content STUDIES ON THE PULMONARY SURFACE-ACTIVE LIPOPROTEIN:
ISOLATION AND CHARACTERIZATION OF ITS PEPTIDE MOIETY
by
J o s e M iguel Jim en ez -S aen z
A D i s s e r t a t i o n P r e s e n te d t o th e
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u l f i l l m e n t o f th e
R eq u irem en ts f o r th e D egree
DOCTOR OF PHILOSOPHY
(B io lo g y )
Ju n e 1968
UNIVERSITY O F S O U T H E R N CA LIFO RN IA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
...................j J A S . f L . M l S . y . E L . . J .l .M E N E 2 „ S . A E N 2 ..................................
under the direction of hJ.S... Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R OF P H I L O S O P H Y
Dean
DISSERTATION COMMITTEE
Chairman
(T yyuU T
TO M Y WIFE MD DAUGHTERS
W H O M ADE A REALITY M Y MOST PRECIOUS DREAM.
TABLE OF CONTENTS
Page
LIST OF TA B LES........................................................................................................... v
LIST OF FIGURES .........................................' ............................ v i
C h a p te r
I . INTRODUCTION . ............................................................................. 1
G e n e r a l and H i s t o r i c a l R eview
S ta te m e n t o f t h e P ro b lem
A cknow ledgem ents
L I I . MATERIALS AND METHODS....................................................... 29
I s o l a t i o n and C h a r a c t e r i z a t i o n o f t h e Long
S u r f a c e - A c t i v e L ip o p r o t e i n Complex
C h em ical C h a r a c t e r i z a t i o n o f t h e S A -L ip o p ro te in
I s o l a t i o n and P u r i f i c a t i o n o f t h e P e p t i d e M o iety
o f t h e S A -L ip o p ro te in
C h em ical C h a r a c t e r i z a t i o n o f t h e S A -P e p tid e
M o iety
P h y s i c a l C h a r a c t e r i z a t i o n o f t h e S A -P e p tid e
M o iety
B i o l o g i c a l A c t i v i t y
I I I . R ESU LTS............................................................. 67
I s o l a t i o n and C h e m ica l A n a ly s is o f
S A -L ip o p ro te in
D e l i p i d a t i o n
I
S o l u b i l i t y o f t h e D e lip id iz e d S A -L ip o p ro te in
P u r i t y of S A -P ep tid e
C hem ical A n a ly s is of S A -P ep tid e
P e p tid e -C a rb o h y d ra te M oiety
Amino A cid A n a ly s is
C om position o f S A -P ep tid e M oiety
P a r t i a l S p e c i f i c Volume
S e d im e n ta tio n C o n s ta n ts o f S A -P ep tid e
M o le c u la r W eight o f S A -P ep tid e
B i o l o g i c a l A c t i v i t y o f S A -L ip o p ro te in and
o f S A -P ep tid e
I V . DISCUSSION ....................................................................................
V.. SUMMARY.........................................................................................
V I. LITERATURE C IT E D ...................................................... . . .
116
132
136
!
LIST OF TABLES
T a b le Page
1. C h em ical C om p o sitio n o f V a rio u s Lung S u r fa c e - A c tiv e
L ip o p r o t e i n s ....................................................................... . . . . . 69
2 . T o t a l C h o l e s t e r o l , P h o s p h o lip id and T o t a l N itro g e n
C o n te n t in 1 ml o f a C o n c e n tr a te d S u sp e n sio n o f Cow
Lung S A -L ip o p ro te in B e fo re and A f t e r D e l i p i d a t i o n
w ith T h ree D i f f e r e n t T e c h n iq u e s .................................................. 71
3. S o l u b i l i t y V alues of t h e D e l i p i d i z e d Cow Lung
S A - L ip o p r o te in ........................................................................................... 73
i 4 . C hem ical C o m p o sitio n o f S A -L ip o p ro te in and
j S A - P e p tid e .................................. 83
I
i 5. E f f e c t o f V a rio u s P u r i f i c a t i o n P ro c e d u re s on
| S i a l i c A cid C o n te n t o f S A - P e p tid e .............................................. 84
! ' ' I
j 6. Amino A cid R e c o v e rie s from S A -P e p tid e j
! H y d r o ly s a te s .............................................. ' . 87 I
7 . C o m p o sitio n and M o le c u la r W eight o f S A -P e p tid e . . . 92
i 8 . I o n i c G roups o f t h e S A - P e p t i d e 95 j
! - i *
| 9 . C a l c u l a t i o n o f t h e P a r t i a l S p e c i f i c Volume o f t h e [
| S A -P e p tid e from i t s C o n s t i t u e n t s . . 9 7 I
; 10. S e d im e n ta tio n V e lo c ity E x p e rim e n t o f S A -P e p tid e }
| C a l c u l a t i o n o f S e d im e n ta tio n C o e f f i c i e n t e s 103 ;
j •
I . \
! 11. S y n t h e t i c B oundary E x p e rim e n t o f S A -P e p tid e j
j C a l c u l a t i o n o f A rea U nder t h e P eak ( C o ) 109 j
j 12. S e d im e n ta tio n E q u ilib r iu m o f S A -P e p tid e . C a lc u la tio n j
! o f AY a t t h e M id -P o in t Xm in S h o r t Columns 113 I
v
LIST OF FIGURES
I
j
jF ig u re
I 1 . C e l l u l o s e A c e t a t e E l e c t r o p h o r e s i s P a t t e r n o f
j S A -P e p tid e . ...................................................................................................
2 . B e h a v io r o f S A -P e p tid e When G e l F i l t e r e d on a
S ephadex G-100 Column o f Medium G ra in ...............................
!
| 3 . A b s o rp tio n S p e c t r a o f S A -P e p tid e ................................................
s
I
; 4 . R e c o v e rie s a s a F u n c tio n o f Time o f H y d r o ly s is f o r
I V a rio u s Amino A c id s and Ammonia, G iven as Grams o f
i . '
j R e s id u e p e r 100 Grams o f S A - P e p tid e ...........................................
!
j- 5. S c h l i e f e n P a t t e r n from a T y p i c a l S e d im e n ta tio n
| V e l o c i t y Run o f S A -P e p tid e , A f t e r 53 M in u te s a t R o to r
Speed o f 5 6 ,1 0 0 r . p . m . ............................................................. .....
! —
| 6 . ' P l o t o f D a ta from a T y p ic a l S e d im e n ta tio n V e lo c ity
I E x p e rim e n t o f S A -P e p tid e , f o r t h e C a l c u l a t i o n o f t h e
S lo p e s o f t h e L in e s f o r E ach One o f t h e Two Main
Com ponents (Slow and F a s t M oving C om ponents) . . . .
7 . D iagram f o r a S h o rt-C o lu m n S e d im e n ta tio n E q u ilib r iu m
E x p e rim e n t, in' Which t h e S c h l i e r e n P a t t e r n s o f
Sam ple and S o lv e n t h av e been S u p erim p o sed ..........................
Page
77
79
81 j
>
i
{
i
i
I
90 i
J
102 j
I
i
!
j
106 ;
112 S
i
v i
CHAPTER I
INTRODUCTION
G e n e r a l
The lu n g a l v e o l i o f mammals, b i r d s , r e p t i l e s , and a m p h ib ia n s
a r e l i n e d w ith a f i l m o f s e m i - s o l i d c h a r a c t e r , h i g h l y i n s o l u b l e in
w a te r , and f r e e l y p e rm e a b le t o g a s e s , w h ich i s c a p a b le o f a lm o s t
a n n u l l i n g t h e s u r f a c e t e n s i o n o f t h e f l u i d s w ith w h ich i t i s in
c o n ta c t-. T h is f i l m i s u n d e r l a i n by a l a y e r o f a l i p o p r o t e i n o f
h ig h m o le c u la r w e ig h t, t h e " l i n i n g com plex" from w h ich t h e f i l m
i s fo rm e d . The p r e s e n c e o f t h i s s u r f a c t a n t a g e n t on t h e s u r f a c e
jof t h e f i l m o f m o i s t u r e c o a t i n g t h e a i r s p a c e s s e r v e s t o s t a b i l i z e
i
[the dynam ic a c t i v i t y o f t h e lu n g s ( C le m e n ts , 1962A; P a t t l e , 1 9 6 5 ),
| From p h y s ic s we know t h a t a t an i n t e r f a c e , t h e a t t r a c t i o n o f
( s u rfa c e m o le c u le s by t h e i r n e i g h b o r s b elo w t h e s u r f a c e i s incom -
j
{ p le te ly c o u n t e r b a la n c e d by a t t r a c t i o n fro m s i m i l a r m o le c u le s above
t h e s u r f a c e . B e c a u se t h e n e t p u l l i s dow nw ard, t h e s u r f a c e s h r i n k s
t o t h e l e a s t p o s s i b l e a r e a . The r e s u l t i n g s u r f a c e t e n s i o n i s a
! j
jfo rc e d i r e c t e d p a r a l l e l t o t h e s u r f a c e an d i s m e a su re d in d y n e s .j
2
p e r c e n tim e te r o f l e n g t h o f s u r f a c e . The f o r c e e x e r t e d in a g iv e n
s u r f a c e i s e q u a l t o tw ic e t h e t e n s i o n d i v i d e d by t h e r a d i u s o f thej
s u r f a c e (G ucker and S e i f e r t , 1 9 6 6 ). By v i r t u e o f t h i s te n d e n c y t o
c o n t r a c t a c u rv ed s u r f a c e , and g iv en t h e t i n y d im e n sio n s o f t h e
a v e r a g e - s i z e a l v e o l u s , c a l c u l a t i o n s h av e shown t h a t t h e s u r f a c e
t e n s i o n o f t i s s u e f l u i d ( a lm o s t 50 d y n es p e r c e n tim e te r ) s h o u ld
f
je x e r t a c o n s id e r a b le f o r c e in a l v e o l i . A c c o rd in g t o C lem ents
j(1962A ), ,ra t 50 dynes p e r c e n t i m e t e r in a s u r f a c e w ith a r a d i u s i
j .
|of o n ly .05 m i l l i m e t e r , i t w ould p ro d u c e a f o r c e o f 2 0 ,0 0 0 dynes -
| ' • !
[per s q u a re c e n tim e te r , w hich e x p r e s s e d as p r e s s u r e , i s equal, t o ;
I ' :
j i
20 c e n tim e te r s o f w a t e r . ” T h e se c a l c u l a t i o n s c e r t a i n l y s u g g e s t j
(
t h a t s u r f a c e t e n s i o n p la y s an im p o rta n t r o l e in t h e p h y s io lo g y j
j
i
o f lu n g s . |
|
s The id e a t h a t s u r f a c e t e n s i o n i s o f im p o rta n c e in lu n g
[dynamics was f i r s t p ro p o se d i n 1929 by K a r l von N e e rg a a rd , o f t h e :
‘ • i
[U n iv e rs ity C l i n i c in Z u r ic h . From h i s own o b s e r v a tio n s in lu n g j
|
m e c h a n ic s, he was a b le t o p r e d i c t ( s e v e r a l y e a rs b e f o r e i t s d i s - j
r i
i - - i
jcovery) t h a t t h e r e s p i r a t o r y u n i t s w ould b e l i n e d w ith a m a t e r i a l j
r I
! . '
jof low s u r f a c e t e n s i o n .(C le m e n ts , 1962A ). L a t e r , Mead, W h itte n - 1
! i
t ;
{ |
jb e rg e r, and R ad fo rd (1954) a r r i v e d a t p r a c t i c a l l y t h e same c o n - ■
t . j
[e lu sio n when s tu d y in g lu n g p h y s io lo g y . W ith t h e s e s u g g e s tio n s j
i i
! ■ i
[in m ind, i n v e s t i g a t o r s began to, lo o k i n t o t h e t i s s u e f l u i d o f t h e j
lu n g f o r t h e p re s e n c e o f a s u r f a c e a c t i v e a g e n t . |
I |
i A t a number o f l a b o r a t o r i e s t h e p re s e n c e o f a s u r f a c t a n t j
a g e n t was soon d e m o n s tra te d in t h e t i s s u e f l u i d e x t r a c t e d from i
t h e lu n g s . In E n g la n d , from s t u d i e s on t h e p r o p e r t i e s o f foam
o b ta in e d from r a b b i t lu n g s , P a t t l e (1955) was a b le t o c o n c lu d e
t h a t t h e lu n g a l v e o l i a r e i n t e r n a l l y c o a te d by an i n s o l u b l e l i n
king f i l m , and t h a t t h e o b s e rv e d s t a b i l i t y o f t h e lu n g f r o t h was
! i
idue t o t h e p r e s e n c e of an i n s o l u b l e s u r f a c e l a y e r on t h e b u b b le s , j
I ‘ |
iThis p r e l im i n a r y r e p o r t was f u r t h e r c o n firm e d by s t u d i e s on t h e I
i !
b e h a v io r o f b u b b le s from sputum o f p a t i e n t s o r a n im a ls w ith p u l - j
| |
rnonary edema ( P a t t l e , 1 9 5 6 ), o r from w ash in g s o b ta in e d when sa lin e *
i
I
!
jwas p o u red i n t o th e t r a c h e a in v i v o , o r a f t e r e x c i s io n o f t h e
jlungs ( P a t t l e , 1 9 5 8 ). The f r o t h o b ta in e d by t h e s e means had
j
jnany p e c u l i a r i t i e s : i t c o u ld be a t t a c k e d by p a n c r e a t i n and by
| , ; ■
jtr y p s in , b u t i t was r e s i s t a n t t o c h e m ic a l a n tif o a m s . S in c e r e -
j. |
d i s t a n c e t o a n tifo a m i s c l o s e l y l i n k e d w ith low s u r f a c e t e n s i o n j
i
I • I
and h ig h s u r f a c e a c t i v i t y ( P a t t l e , 1 9 5 0 ), t h e s u r f a c e t e n s i o n of j
I i
t h e f r o t h b u b b le s was t h e r e f o r e i n v e s t i g a t e d . From t h e s e s t u d i e s ;
i ;
Lt was c o n c lu d e d t h a t t h e m a t e r i a l r e s p o n s i b le f o r t h e low s u r - j
f a c e t e n s i o n o f t h e lu n g s i s a m onom olecular f i l m w hich l i n e s t h e j
I I
f e s p i r a t o r y u n i t ; f u r th e r m o r e , t h a t t h e l i n i n g l a y e r o f t h e j
b u b b le s i s form ed by t h e a l v e o l a r c e l l s , and t h a t t h i s m a t e r i a l |
i J 3 I
i s p r i n c i p a l l y a l i p o p r o t e i n c o n ta in in g la r g e amounts o f - l e c i t h i n
( P a t t l e , 1955, 1956, 1958, 1963).
j
i S im ila r s tu d i e s were done in t h e U n ited S t a t e s by Clem ents
!
i
land h i s c o lle a g u e s w ith s i m i l a r f in d in g s (C lem en ts, 1956, 1957;
t . •
| •
'C lem ents, e t , a l . , 1958; Brown, e t a l . , 1 9 59). By u t i l i z i n g a
m o d ified W ilhelmy b a la n c e f o r m easurem ents o f s u r f a c e t e n s i o n ,
i t was shown t h a t th e s u r f a c e te n s io n of lu n g e x t r a c t s d e c re a se d
g r e a t l y when th e s u r f a c e a r e a was com pressed. T h is was found to
i '
jbe an im p o rta n t c h a r a c t e r i s t i c of th e pulm onary s u r f a c t a n t
[(C lem ents, 1 9 58). I t was e v id e n t t h a t i f such a d e c re a s e in s u r
f a c e te n s io n w ith d e c re a s e in s u r f a c e a r e a o c c u rre d in th e lu n g ,
'
th e s m a ll a l v e o l i would have low s u r f a c e te n s io n and l e s s p r o
p e n s ity t o c o l l a p s e , w hereas l a r g e r a l v e o l i would have g r e a t e r
[su rface te n s io n w hich would r e s i s t f u r t h e r e n la rg e m e n t. T h is
[ e ffe c t would s t a b i l i z e th e a l v e o l i and p ro v id e f o r a ran g e o f
jsm all a l v e o l a r s iz e s (C lem en ts, 1962A, 1962B ).
| Up t o now i t i s c l e a r t h a t two groups o f i n v e s t i g a t o r s have
a r r i v e d a t s im i l a r c o n c lu s io n s and have c l e a r l y e s ta b l i s h e d th e
e x is te n c e of a s u r f a c e a c t i v e a g e n t which i n t e r n a l l y c o a ts th e
i
jlung a l v e o l i .
5
P h y s i o l o g i c a l F u n c tio n s o f t h e S u r f a c e A c tiv e L i p o p r o t e i n Complex
I
; E a r l y in t h i s c h a p t e r i t was m e n tio n e d t h a t t h e lu n g s u r - j
f a c t a n t a g e n t s e r v e s t o s t a b i l i z e t h e dynam ic a c t i v i t y o f t h e
lu n g s . E x p la in in g t h e d i f f e r e n t f u n c t io n s o f t h e s u r f a c e a c t i v e
j l i p o p r o t e i n com plex o f t h e lu n g s , C lem en ts (1962A) h as s t a t e d
J
[th a t t h i s s u b s ta n c e a c t s t o e q u a l i z e t h e t e n s i o n in t h e a i r s p a c e s
jas th e y expand and c o n t r a c t . He s u g g e s ts t h a t in an exp an d ed a i r
i . |
jspace t h e l a y e r o f s u r f a c t a n t a g e n t i s s u p p o s e d ly a t t e n u a t e d and
< I
i \
; • i
jas a r e s u l t of t h i s , s u r f a c e t e n s i o n i s i n c r e a s e d a c c o r d i n g l y . j
I !
However, by t h e i n c r e a s e in t h e r a d i u s o f t h e a i r s p a c e , t h e ;
i n c r e a s e i n t h e t e n s i o n i s p a r t i a l l y o f f s e t . As t h e a i r sp a c e
i
!
c o n t r a c t s , t h e i n c r e a s i n g c o n c e n t r a t i o n o f t h e s u r f a c t a n t a g e n t j
j
r e d u c e s t h e s u r f a c e t e n s i o n , a g a in d e c r e a s i n g t h e p r e s s u r e in ;
'th e a i r s p a c e . A ssum ing t h a t t h e c o n c e n t r a t i o n o f s u r f a c e a c t i v e ;
la g e n t i s d i f f e r e n t in s m a l l e r and in l a r g e r a l v e o l i , C lem ents
i
was a b le t o e x p la in how in t h e i n t e r f a c e o f t i s s u e f l u i d and a i r j
i
i
t h e p r e s s u r e i s h o m ogeneously d i s t r i b u t e d , so t h a t a l l th e a l v e o l i ;
• !
may rem ain open and t h e i r f u n c t i o n can be a c c o m p lish e d sm o o th ly j
and c o o r d i n a t e d . i
i !
I The s u r f a c t a n t l i n i n g h as a se c o n d no l e s s im p o r ta n t f u n c - j
• 5
t i o n r e l a t i n g t o o sm o tic f o r c e s a c t i n g a c r o s s t h e s u r f a c e o f t h e j
I - !
jLungs. T h is f u n c t io n h a s been e x p la in e d by s a y in g t h a t t h e h ig h
i j
I _____________________________ __;_ ;
6
m o le c u la r w e ig h t an d s e m i-p e rm e a b le c h a r a c t e r o f t h e l i p o p r o t e i n
com plex k e e p s t h e f i l m o f m o i s t u r e on t h e s u r f a c e from d ra w in g
: f l u i d i n t o t h e a i r s p a c e s (C le m e n ts , 1962A; P a t t l e , 1965; B o n d u r-
;ant and Giammona, 1965) . i
S u r f a c e A c tiv e L i p o p r o t e i n Complex and P a th o lo g y
A l t e r e d s u r f a c e c h a r a c t e r i s t i c s o f lu n g e x t r a c t s h a v e b een i
fo u n d i n s e v e r a l c l i n i c a l c o n d i t i o n s . A b n o rm a lly h ig h s u r f a c e
t e n s i o n s o f lu n g e x t r a c t s o f f e t u s e s and o f i n f a n t s w ith h y a l i n e
membrane d i s e a s e h a v e b een r e p o r t e d by A very and Mead ( 1 9 5 8 ) , and :
by A v ery ( 1 9 6 2 ) , who h av e s u g g e s te d t h a t t h i s d i s e a s e , c h a r a c t e r
i z e d by a t e l e c t a s i s , m ig h t be due in p a r t t o a b n o r m a lity o f s u r - ;
f a c t a n t . I t h as b een a l s o shown by G ru e n w a ld , e t _al. ( 1 9 6 2 ), j
t h a t t h e lu n g s o f i n f a n t s w ith h y a l i n e membrane d i s e a s e had a
lo w s u r f a c t a n t in d e x and d e f i c i e n t s t a b i l i t y . I n v e s t i g a t i o n s o f
P a t t l e and a s s o c i a t e s ( 1 9 6 2 ), and m ore r e c e n t l y t h e w ork o f s
i ;
i !
R e y n o ld s and c o l l a b o r a t o r s ( 1 9 6 5 ) , have sh o w n ;t h a t in c a s e s o f |
r e s p i r a t o r y d i s e a s e syndrom e o f t h e new born t h e lu n g s w ere i n c a p - ;
a b l e o f fo rm in g a n o rm a l l i n i n g f i l m b e c a u s e o f a d e f i c i e n c y o f
t h e l i p o p r o t e i n c o m p lex , from w h ich t h e f i l m i s fo rm e d . From
t h e s e f i n d i n g s , i t was c o n c lu d e d by R e y n o ld s and c o l l e a g u e s ( 1 9 6 5 ) j
| j
t h a t t h e so c a l l e d "m em brane", w h ich a p p e a r s i n t h i s d i s e a s e , i s a!
7
se c o n d a ry f e a t u r e a r i s i n g from th e l u n g ’s i n a b i l i t y t o form a
n orm al l i n i n g f il m , and c o n s t i t u t e d by t r a n s u d a t e drawn from th e
b lo o d .
T hese s t u d i e s have been co n firm ed in s e v e r a l o th e r la b o r a - j
t o r i e s and to d a y th e y c o n s t i t u t e s tr o n g e v id e n c e in su p p o rt of
rthe g e n e r a lly a c c e p te d h y p o th e s is t h a t f a i l u r e t o form th e n o r - |
|
m al l i n i n g com plex m ight be in v o lv e d in c a u s a tio n o f t h e d is e a s e !
( P a t t l e , 1 9 6 5 ). '
I t has been found by Brown (1 9 6 5 ), t h a t t h e s u r f a c e t e n s io n ;
iand co m p o sitio n o f emphysematous lu n g e x t r a c t s a r e s i m i l a r t o
ithose o f n orm al lu n g s . However, w ith pneum onia o r pulm onary edema:
| ;
;p re s e n t a t d e a th t h e s u r f a c e a c t i v i t y of t h e lu n g e x t r a c t s ' i s
^usually d im in is h e d . j
E a r ly in 1964, Abrams and T a y lo r (1 9 6 4 ), and T a y lo r and
Abrams (1 9 6 4 ), found t h a t th e s u r f a c e a c t i v e l i p o p r o t e i n e x tra c te d :
ifrom th e lu n g s of r a b b i t s , lam bs, and human b e in g s s tr o n g ly i n - i
i !
i , :
i . i
jh ib its c l o t r e t r a c t i o n and a l s o th e f i b r i n o l y t i c sy stem . Con- j
v e r s e l y , th e y a l s o found t h a t f ib r in o g e n i n h i b i t s s u r f a c e a c t i v i t y
p f th e l i p o p r o t e i n complex in m o lar r a t i o s o f 1 :1 . From t h e s e j
and o t h e r f i n d i n g s , Abrams (1 9 6 6 ), and T a y lo r and Abrams (1966) |
; I
have p ro p o sed a new h y p o th e s is o f t h e p a th o g e n e s is o f pulm onary j
P t e l e c t a s i s and th e h y a lin e membrane in t h e r e s p i r a t o r y d i s t r e s s j
^syndrome o f t h e new born i n v o l v i n g i n t r a - a l v e o l a r (1) n e u t r a l i z a
t i o n o f t h e s u r f a c e a c t i v i t y o f t h e l i p o p r o t e i n by f i b r i n o g e n ,
(2 ) a c t i v a t i o n o f t h e c o a g u l a t i o n enzyme s y ste m , and (3 ) i n h i b i
t i o n o f t h e f i b r i n o l y t i c enzyme sy ste m o f t h e p lasm a t r a n s u d a t e , i
; I
: * i
[The p ro p o se d h y p o th e s is o f Abrams and T a y lo r seems t o a g re e w ith j
jthe p r e v io u s i d e a s u g g e s te d by R e y n o ld s , e t a l . ( 1 9 6 5 ) , t h a t t h e
"membrane" in t h i s d i s e a s e i s r e a l l y a s e c o n d a ry c h a r a c t e r i s t i c j
[a p p e a rin g a s a c o n se q u e n c e o f an a b n o r m a lity r e l a t e d t o t h e s u r -
f a c t a n t a g e n t . N e i t h e r R e y n o ld s , e t a l . (1 9 6 5 ), n o r Abrams and j
'T a y lo r (1966) h y p o th e s iz e d on t h e p o s s i b l e g e n e s is o f t h e s u r -
i ■ '
; f a c t a n t a b n o r m a lity .
| - i
| ' Numerous s ta t e m e n t s h a v e b een made in r e l a t i o n s h i p t o t h e j
i • . i
• , l
if a c t t h a t c e r t a i n p a t h o l o g i c a l c o n d i t i o n s o t h e r th a n t h e r e s p i r a -
|
;to ry d i s t r e s s syndrom e o f t h e new born may a r i s e from a b n o r m a lity j
I in t h e s u r f a c t a n t a g e n t . T h u s, f o r ex am p le, i t h a s b een fo u n d
! I
[th a t t h e a p p l i c a t i o n o f to b a c c o smoke t o aqu eo u s s u r f a c e s and t o
i
i
jlung e x t r a c t s c a u s e s ch an g es i n s u r f a c e t e n s i o n . A ls o , t h a t t h e
I
[su rfa c e t e n s i o n o f e x t r a c t s o f lu n g s o f a n im a ls e x p o se d t o c i g a r - j
! i
i
e t t e smoke in l i f e d i f f e r s from t h a t o f n o rm a l a n im a ls ( M i l l e r j
jand B o n d u ra n t, 1 9 6 2 ). C i g a r e t t e smoke was found t o c a u se an i n - j
t [
jc re a se in s u r f a c e c o m p r e s s i b i l i t y of t h e f i l m and t h i s .would be \
* • *
j r e la te d w ith p o s s i b l e ch an g es i n t h e s t a b i l i t y o f t h e lu n g a l v e o l i ;
9
In 1963 C o l l i e r found t h a t e x t r a c t s of m inced lu n g t i s s u e ■
from r a b b i t s t h a t had d ie d a f t e r e x p o su re t o 100 p e r c e n t C >2 a t
one a tm o sp h e re , d i f f e r e d in s u r f a c e t e n s io n as compared t o lu n g
e x t r a c t s from norm al r a b b i t s . C o l l i e r (1963) found h ig h s i g n i f i - j
j
c a n t d i f f e r e n c e s (P < 0 .0 1 ), and he b e lie v e d t h a t th e ab sen ce in . j
; s u r f a c e a c t i v i t y o f t h e e x t r a c t s may be an im p o rta n t cau se o f >
pulm onary damage in oxygen p o is o n in g . T hese r e s u l t s have been ’ j
c a r e f u l l y co n firm ed by many w o rk e rs. T hus, f o r exam ple, C a ld w e ll !
i
and c o lle a g u e s (1965) have found a l o s s o f s u r f a c e a c t i v i t y of
:lung e x t r a c t s a s s o c i a t e d w ith s e v e re pulm onary damage in dogs
jwhich were exposed t o p ro lo n g e d b r e a th in g o f 98 p e r c e n t of O2 a t :
> !
lone a tm o sp h e re . 'A lm ost s im u lta n e o u s ly , Morgan and c o l l a b o r a t o r s j
l(1965A) r e p o r te d t h a t e x p o s u re t o p u re oxygen p ro d u ces p ro g re s siv e !
! 1
.r e s p i r a t o r y d i s t r e s s , pulm onary edema, and in c r e a s e d pulm onary
;su rfa c e t e n s i o n (w hich r e f l e c t s a l o s s in s u r f a c t a n t a g e n t ) .
M o reover, th e y a l s o r e p o r te d (1965B) h a v in g found t h a t t h e l i p i d i
j
[components o f th e s u r f a c e a c t i v e lip o p r o te in 'c o m p le x , were a lte re d !
a n d t h e e s t e r i f i e d f a t t y acids" in t h e l e c i t h i n f r a c t i o n showed a
jre d u c tio n in p a lm ita te and t o t a l s a t u r a t e d f a t t y a c i d s . The j
i 1
j
je le c tro n m icro g rap h s of t h e lu n g s a f t e r oxygen ex p o su re' showed j
i !
: !
t h i c k e n i n g o f th e a l v e o l a r basem ent membrane and a l t e r a t i o n s in j
i '
t h e s t r u c t u r e of th e la m e l l a r b o d ie s of t h e ty p e I I a l v e o l a r j
e p i t h e l i a l c e l l s . F i n a l l y , Kennedy (1966) o b se rv e d t h a t in 80%
o f t h e a n im als w hich d ie d a f t e r b r e a t h i n g Oj a t two a tm o sp h e re s,
up t o 18 h o u r s , lu n g e x t r a c t s had sabnorm al s u r f a c e t e n s i o n s .
:These s tu d i e s have shown t h a t in oxygen p o is o n in g , an a l t e r e d sur-j
f a c e t e n s i o n o f lu n g e x t r a c t s i s c o n s i s t e n t l y fo u n d . T h is su g -
i
■gests t h a t h ig h oxygen p r e s s u r e s d e n a tu r e t h e s u r f a c e a c t i v e lip o -;
| !
p r o t e i n , e i t h e r d i r e c t l y o r by a c tio n on t h e c e l l s p ro d u c in g i t . j
;Such a p rim a ry e f f e c t co u ld le a d t o many o f th e o th e r p a t h o l o g i c a l
e f f e c t s o b s e rv e d , su ch as r e s p i r a t o r y d i s t r e s s and edema. i
I n h a l a t i o n of a tm o s p h e ric p o l l u t a n t s and some i n d u s t r i a l i
jchem icals can a l s o a f f e c t a l v e o l a r s u r f a c e s , p ro d u c in g l a r g e
i • ;
'amounts o f f r o t h in t h e a ir w a y s , edema, and d e s tr u c t i o n of th e I
jsu rfa c e a c t i v e m a t e r i a l ( M i t t l e r , 1958; P a t t l e , 1965; M endenhall |
; J
'and S to k in g e r , 1962) . W orking w ith s a l i n e w ashings o f mouse
lu n g s exposed t o o zo n e, M endenhall and S to k in g e r (1962) found
i
t h a t d e s t r u c t i o n o f t h e s u r f a c e a c t i v e m a t e r i a l was acco m p lish ed ' ;
i i
' !
|by ozone, and t h a t t h e o b se rv e d a t e l e c t a s i s and pulm onary edema ;
were produced as a co n seq u en ce o f su ch d e s t r u c t i o n . T h e .a u th o r s
jeon elu d e d t h a t t h e s u r f a c t a n t m a t e r i a l may a c t as a p r o t e c t o r of j
ithe a l v e o l a r w a ll a g a i n s t low l e v e l s o f o z o n e . M endenhall and
S to k in g e r a l s o p o in te d o u t t h a t in c r e a s e d p r e s s u r e o f t h e f il m in
!
; i
| - I
th e a l v e o l i may c a u se emphysema. P r e v io u s ly , M acklin (1954) had j
1 1
a l s o s u g g e s te d a r e l a t i o n s h i p b etw een t h e lu n g l i n i n g f i l m and
emphysema.
From t h e f o r e g o i n g d i s c u s s i o n , i t i s c l e a r t h a t o u r p r e s e n t
u n d e r s ta n d in g o f t h e a c t i o n o f a i r p o l l u t a n t s on t h e l i n i n g f i l m
.is r e l a t i v e l y s c a n t y . Much w ork i s in p r o g r e s s and h a s n o t been
!f u l l y r e p o r t e d . T h is a u t h o r b e l i e v e s t h a t t h e e f f e c t s o f a i r
p o l l u t a n t s on lu n g p ro b le m i n v o l v i n g t h e s u r f a c e a c t i v e l i p o -
I p r o t e i n com plex may be p ro n o u n c e d an d m e r i t i n t e n s i v e i n v e s t i g a
t i o n .
^ A p p e a ra n c e , O r i g i n , and C o m p o sitio n o f t h e S u r f a c e A c tiv e
jL i p o p r o t e i n Com plex
! I t h a s b een e s t a b l i s h e d t h a t m a tu re f e t a l lu n g h a s a l i n i n g j
f i l m r e s e m b lin g t h a t o f n o rm a l a d u l t lu n g . E a r l y f e t a l lu n g d o es
h o t fo rm su ch a f i l m ( P a t t l e , 1 9 6 5 ).
C a r e f u l s t u d i e s h a v e shown t h a t t h e c a p a c i t y t o form a l i n - j
i i
|in g f i l m i s c o r r e l a t e d w ith t h e h i s t o l o g i c a l d e v e lo p m e n t o f mouse,j
r a t , and g u in e a p i g s ( P a t t l e , 1 9 6 1 ). The e a r l y f e t a l lu n g i s ;
i
I i
!l i n e d by a c o n tin u o u s l a y e r o f c u b o i d a l e p i t h e l i u m w h ich l a t e r in )
jd ev elo p m en t a p p e a r s t o d e s q u a m a te . When t h i s p r o c e s s b e g i n s , t h e
,a b i l i t y t o fo rm a l i n i n g f i l m a p p e a r s q u i t e s u d d e n ly . B uckingham
( 1 9 6 1 ) , and B uckingham and A v ery (1 9 6 2 ) h a v e fo u n d t h a t t h e
12 j
c h a r a c t e r i s t i c s u r f a c e a c t i v i t y a p p e a rs in e x t r a c t s o f f e t a l lu n g s :
o n - th e e i g h t e e n t h d a y . On t h e o t h e r h a n d , i t h as been found t h a t
in t h e human f e t u s d esq u am atio n o f c u b o id a l e p i th e liu m t a k e s place;
:in t h e lu n g s betw een t h e t w e n t y - f i r s t and t w e n t y - f o u r t h week o f !
I n t r a u t e r i n e d ev elo p m en t ( S h o r t, 1 9 5 0 ), and f e t u s e s o f tw e n ty -
jfiv e weeks have n o rm a l l i n i n g f i l m , a lth o u g h much c u b o id a l e p i -
|
th e liu m i s a l s o found ( P a t t l e , e t . a l . , 1 9 6 2 ). T hese f a c t s su g - !
Igest t h a t in man t h e a b i l i t y t o form, a l i n i n g f i l m d e v e lo p s more
g r a d u a l l y d u r in g t h e f i f t h t o s e v e n th m onths o f g e s t a t i o n ( C le
m e n ts, 1962; P a t t l e , 1 9 6 5 ).
A l l t h e e v id e n c e th u s f a r p o i n t s t o an o r i g i n f o r t h e l i n
i n g f i l m com plex in t h e a l v e o l a r ' r e g io n o f t h e lu n g by t h e i
i
! T T a l v e o l a r c e l l s " ? w hich s in c e 1954 have been a t t r i b u t e d as h a v in g j
• i
a s e c r e t o r y f u n c tio n and b e in g c a p a b le o f r e g u l a t i n g s u r f a c e t e n - I
s io n (M a c k lin , 1 9 5 4 ).
; K la u s , e t . a l . (1962) s u p p o rt t h e h y p o th e s is t h a t th e s u r -
i
jface a c t i v e l i n i n g o f t h e lu n g d e v e lo p s d u r in g t h e p r o c e s s o f
lame l i a r t r a n s f o r m a t i o n o f m ito c h o n d r ia ” in t h e a l v e o l a r e p i
t h e l i a l c e l l . N e v e r t h e l e s s , d i r e c t e v id e n c e o f s e c r e t i o n o n to
I 7
t h e s u r f a c e was n o t o b ta in e d . O th e r i n v e s t i g a t o r s , on t h e co n -
j
t r a r y , have s u g g e s te d t h a t s in c e t h e com plex h a s ”a d e f i n i t e
! . i
; i
S t r u c t u r e " r e l a t e d t o t h a t o f t h e c e l l membrane, and e a r l y
13
e l e c t r o n m ic ro g ra p h s o f lu n g t i s s u e h a v e shown a s t r u c t u r e l e s s
[ " s u r f a c e m em brane” l i n i n g t h e a l v e o l a r e p i t h e l i u m (C h a s e , 1 9 5 9 ),
;i t was th o u g h t t h a t i t m ig h t b e p ro d u c e d c o n t i n u o u s l y fro m t h e
c e l l s u r f a c e membrane (M e n d e n h a ll and Sun, 1 9 6 4 ). j
R e c e n tly a s e r i e s o f i n v e s t i g a t i o n s h a v e b een c a r r i e d o u t
jin o r d e r t o c l a r i f y f u r t h e r t h e s i t e o f o r i g i n o f t h e s u r f a c e •']
■ i
i a c tiv e m a t e r i a l . F o r e x a m p le , H ackney and c o l l e a g u e s (1 9 6 5 ) h a v e 1
'been a b l e t o o b s e rv e t h a t when f r o z e n s e c t i o n s o f u n f ix e d r a b b i t
[lung t i s s u e w ere v iew ed w ith an u l t r a v i o l e t m ic ro s c o p e , a l v e o l a r j
t i s s u e b u b b le s showed a b r i g h t f l u o r e s c e n t l i n i n g t h a t s t a i n e d
w i t h l i p i d - b i n d i n g flu o r o c h r o m e d y e s . The a p p e a r a n c e , c h e m ic a l
[ r e a c t i o n , a u t o f l u o r e s c e n c e , and flu o r o c h r o m e s t a i n i n g o f t h e s e j
\ _ i
* !
j t i s s u e s e c t i o n s p ro m p te d t h e i d e a t h a t a d e t a c h a b l e s u r f a c e a c tiv e !
i *
T i p i d s u b s ta n c e l i n e s t h e a l v e o l i and i s fo rm ed i n a l v e o l a r c e l l s . ;
U t i l i z i n g a d i f f e r e n t a p p r o a c h , h i s t o c h e m i c a l s tu d y o f t h e m e ta b o -
■ ' '
ilism o f a l v e o l a r c e l l s , S a i d , e t a l . ( 1 9 6 6 ) h a v e fo u n d t h a t t h e j
' ‘
[ la r g e r a l v e o l a r c e l l s o f n o rm a l lu n g a r e r i c h in enzym es concerned!
w ith o x i d a t i v e and b i o s y n t h e t i c p a th w a y s . In s i t u a t i o n s w here |
[ a b i l i t y o f t h e lu n g t o p ro d u c e s u r f a c t a n t was im p a i r e d , t h e enzyme]
i !
r i c h c e l l s w ere l a c k i n g o r a b s e n t t o o . T hey c o n c lu d e d t h a t t h e s e j
enzym es may be i n v o lv e d in s y n t h e s i s o f t h e s u r f a c e a c t i v e l i p o - |
p r o t e i n co m p lex . B uckingham , e t a l . ( 1 9 6 6 ) , s tu d y i n g p h o sp h o - !
i
!
l i p i d s y n t h e s i s in t h e l a r g e p u lm o n a ry c e l l in r e l a t i o n - t o lu n g
1 4
s u r f a c t a n t , o b ta in e d v ery i n t e r e s t i n g f in d i n g s . They i n v e s t i
g a te d th e d i s t r i b u t i o n of r a d i o a c t i v i t y in t h e l i p i d f r a c t i o n s of
r a b b i t lu n g and l i v e r f i f t e e n t o n i n e t y m in u te s a f t e r in tra v e n o u s ,
a d m in is tr a tio n of a c e t a t e and p a l m i t a t e . The s p e c i f i c a c ti- i
i
v i t y of p a l m i t a t e was h a l f as h ig h in lu n g as in l i v e r . Lung
[p a lm ita te was r a p i d l y i n c o r p o r a te d i n t o p h o s p h o lip id s (85%), p r i - j
m a r ily l e c i t h i n (60%). Lung t i s s u e was tw ic e as a c t i v e as l i v e r I
*2
in i n c o r p o r a t i n g H a c e t a t e i n t o l i p i d s . A c e ta te was in c o r p o r a te d
r a p i d l y i n t o lu n g p h o s p h o lip id s (90%), e s p e c i a l l y i n t o l e c i t h i n ,
1(60%). A u to ra d io g ra p h s of lu n g d e m o n stra te d l a b e l i n g of th e !
cy to p lasm o f th e l a r g e a l v e o l a r c e l l s : b etw een , b u t n o t i n s i d e :
[of t h e i r c h a r a c t e r i s t i c c y to p la s m ic i n c l u s i o n s . T h is o b s e r v a tio n i
i !
; I
ileav es l i t t l e d oubt t h a t th e cy to p la sm of a l v e o l a r c e l l s i s an |
j j
[im p o rtan t s i t e o f p h o s p h o lip id tu r n o v e r in lu n g , and c o n firm s t h e [
o b s e r v a tio n s made by Tom bropoulos (1964) t h a t in lu n g , f a t t y a c id [
;s y n th e s is i s m ost a c t i v e in th e m i t o c h o n d r i a l - r i c h s u b c e l l u l a r j
j ' !
[ f r a c tio n s , and t h a t t h e tu r n o v e r os p h o s p h o lip id s , e s p e c i a l l y j
l e c i t h i n , i s e x tre m e ly r a p i d . The a u th o r s have i n f e r r e d t h a t t h e ;
'h ig h tu r n o v e r r a t e o f lu n g l e c i t h i n i s lin k e d t o fo rm a tio n and
: i
i >
[d isch arg e o f s u r f a c t a n t d i p a l m i t y l l e c i t h i n . i n t o th e a l v e o l a r j
i ' i
| j
.•space. |
j ■ . i
I Niden (1967) has found t h a t t h e n o n c i l i a t e d b r o n c h io la r i
15
c e l l s ( C la r a c e l l s ) l i n i n g t h e t e r m i n a l a irw a y s a c t i v e l y s e c r e t e
a p h o s p h o lip id . In c o n t r a s t , t h e l a r g e a l v e o l a r e p i t h e l i a l c e l l s
( ty p e I I , g r a n u la r pneum ocyte) a r e a c t i v e p h a g o c y tic c e l l s . He
p ro p o se d t h a t t h e C la r a c e l l i s the. main s o u rc e o f pulm onary p h o s
p h o l i p i d p r o d u c tio n (p re su m a b ly s u r f a c t a n t ) w h ile t h e la rg e , alveo-
j l a r c e l l i s r e s p o n s i b le f o r i t s s u b se q u e n t breakdow n.
A v e ry r e c e n t r e p o r t o f one o f t h e i n v e s t i g a t o r s of th e
^ U n iv e rsity o f S o u th e rn C a l i f o r n i a , Evans (1 9 6 8 ), who h as been
s tu d y in g c e l l u l a r re n e w a l in th e pulm onary a l v e o l i of t h e mouse
and r a t , made t h e o b s e r v a tio n t h a t t h e e n d o t h e l i a l c e l l s l i n i n g
c a p i l l a r i e s o f pulm onary a l v e o l i a r e c a p a b le o f c e l l d i v i s i o n
jw ith a t u r n o v e r tim e of t e n t o tw e lv e d a y s . The a l v e o l a r w a ll
I c e l l s h av e a t u r n o v e r tim e o f tw e n ty t o t w e n t y - f i v e d a y s . T hese
b i n d i n g s s u p p o rt t h e id e a t h a t n o t o n ly a r e th e a l v e o l a r c e l l con
j s t i t u e n t s m e ta b o liz e d v e ry r a p i d l y , b u t t h e c e l l s a r e renew ed
r a p i d l y a s w e l l .
| ■ ;
| F i n a l l y , t h e r e i s t h e r e c e n t work o f a g ro u p o f i n v e s t i g a -
i ‘
t o r s l e d by W etton and C avagna (W etto n , e t a l . , 1967; and
jCavagna, e t a l . , 1 9 6 7 ), who have exam ined r e l a t i o n s h i p s betw een
i '
i
j s u r f a c t a n t a g e n t and pulm onary c e l l s . I t h as been found by
i
j ' '
W etton and c o lle a g u e s (1967) t h a t o n ly t h o s e f r a c t i o n s o f lu n g
jwashings from l i v i n g dogs w hich c o n ta in e d c e l l s w ere a b l e t o
lo w er t h e s u r f a c e t e n s i o n , and th o s e w ith o u t c e l l s d id n o t .
Cavagna, e t a l . (1 9 6 7 ), w orking on t h e same p ro b le m , have shown
t h a t t h e s e lu n g c e l l s , when washed and shaken in s a l i n e , lo s e t h e j
s u r f a c e - a c t i v e p r o p e r ty , b u t i t re -a p p e a re d in t h e c e l l w ashes. I
i
The s u r f a c t a n t th u s removed co u ld be re a d s o rb e d on washed c e l l s
'o b ta in e d from lu n g o r on washed w h ite c e l l s o b ta in e d from b lo o d j
by a llo w in g t h e c e l l s and s u r f a c t a n t t o s ta n d in c o n ta c t o v e r- j
n i g h t . I t was a l s o o b se rv e d t h a t lu n g c e l l s a lo n e o r t h e i r mem
b r a n e s and p a r t i c l e s o b ta in e d by ”s o n i c a t i o n n l a c k th e s u r f a c e - ■
ia c tiv e p r o p e r t y . T hus, i t a p p e a rs t h a t t h e s u r f a c t a n t l i p o p r o t e i n
com plex i s an autonom ous e n t i t y w hich n o rm a lly e x i s t s in an a d - ;
is o rb e d s t a t e on t h e s u r f a c e s of lu n g c e l l s . j
! • i
i From t h e f o re g o in g e x p o s it i o n , i t i s c l e a r t h a t , w ith th e i
I i
^exception o f th e works o f K la u s, e t a l . (1 9 6 2 ), and M endenhall
and Sun (1 9 6 4 ), no one h as t r i e d a d i r e c t a p p ro ac h t o s o lv e t h e
jproblem o f t h e s i t e o f o r i g i n o f th e w hole s u r f a c e a c t i v e l i p o - j
j • |
jp ro te in com plex. I t i s b e lie v e d t h a t one o f t h e re a s o n s f o r t h i s j
i s b e c a u se t h e c h e m ic a l co m p o sitio n o f t h i s l i p o p r o t e i n i s n o t j
'f u l l y known y e t . We know t h a t p r o t e i n s a r e s y n th e s iz e d by t h e \
i
iendoplasm ic r e tic u lu m and t h a t l i p i d s may be s y n th e t i z e d e i t h e r
i
' ■ i
i n t h e c y to p la sm o r in m ito c h o n d ria , b u t t h i s does n o t s o lv e t h e j
i !
p ro b lem o f t h e g e n e s is o f t h i s l i p o p r o t e i n . A re t h e s y n th e s is o f j
| i
■ ■ f
t h e l i p i d and p e p tid e m o ie tie s o f t h i s l i p o p r o t e i n c a r r i e d o u t J
17
s im u lta n e o u s ly by t h e same c e l l s ? When and w here does t h e b in d in g
o f l i p i d and p e p t i d e m o ie tie s o ccu r? Thus f a r , we know so m eth in g
a b o u t t h e s y n t h e t i c s i t e o f some o f t h e l i p i d com ponents o f t h e ;
i
l i p i d m o ie ty of t h e s u r f a c t a n t . V i r t u a l l y n o th in g i s known ab o u t |
; |
‘t h e p e p t i d e m o ie ty .
C h em ical a n a l y s i s h as shown t h a t t h e s u r f a c t a n t a g e n t o f j
• i
;t h e lu n g i s a l i p o p r o t e i n com plex. The f i r s t a tte m p t t o e s t a b l i s h
' i t s n a t u r e was done by P a t t l e and Thomas (1 9 6 1 ). Cow. lu n g w ash
in g s w ere s p e c t r o p h o t o m e t r ic a l ly a n a ly z e d in an i n f r a - r e d a b s o rp - j
' ! . i
^tion s p e c tro p h o to m e te r and a l a r g e am ount o f l e c i t h i n a s s o c i a t e d
i i
I w ith p r o t e i n was fo u n d . K la u s , e t a l . (1 9 6 1 ), u t i l i z i n g t h e J
m ethod s u g g e s te d by B o n d u ran t (1960) f o r t h e i s o l a t i o n o f t h e j
s u r f a c e - a c t i v e m a t e r i a l , found t h a t i t c o n ta in e d 50-70 p e r c e n t j
‘l i p i d s and 5 p e r c e n t n i t r o g e n (32% p r o t e i n ) . S e p a r a tio n o f t h e
imajor l i p i d f r a c t i o n s on s i l i c i c a c i d colum ns y ie ld e d 74% p h o s
p h o l i p i d s , 8% c h o l e s t e r o l , 10% t r i g l y c e r i d e s , 8% f a t t y a c i d s and j
j e s s e n t i a l l y no c h o l e s t e r o l e s t e r s . T hese s g u d ie s have been con- I
firm e d by F i n l e y , e t . _al. (1962) . When l i p i d e x t r a c t s from dog j
lu n g w ash in g s w ere i d e n t i f i e d by c h e m ic a l and c h ro m a to g ra p h ic !
I
i
a n a l y s i s , i t was fo u n d t h a t l i p i d d i s t r i b u t i o n was s i m i l a r t o j
! . i
! . 1
jth a t o f e x c is e d b e e f lu n g edema f l u i d d e s c r ib e d by K la u s , e t ja l. |
1(1961) . A h ig h c o n te n t o f l e c i t h i n in f l u i d c o l l e c t e d fro m e x - j
! j
Icised lu n g was a l s o r e p o r t e d by Brown (1 9 6 2 ). Adams and F u iiw a ra 1
18
(1963) found t h a t p h o s p h o lip id p re d o m in a te s i n th e l i p i d s o f th e
t r a c h e a l f l u i d o f f e t a l lam bs; and Morgan and c o lle a g u e s (1965B)
have i d e n t i f i e d and compared l i p i d s o b ta in e d from t h e a l v e o l a r
s u r f a c e by la v a g e w ith th o s e o b ta in e d from a hom ogenate of w hole I
5 i
;lung. I t was fo u n d t h a t o n ly one l i p i d com ponent, p h o s p h a tid y l-N ,;
jN -d im eth y le th a n o la m in e , c o n s i s t e n t l y p ro d u c e s m arked s u r f a c e t e n - I
s io n lo w e rin g and was shown t o be a b o u t 6% o f b o th a l v e o l a r and !
w hole lu n g t o t a l l i p i d .
! Many s t u d i e s on t h e s u r f a c e a c t i v e l i p o p r o t e i n have been |
c a r r i e d o u t u t i l i z i n g t h e i s o l a t i o n te c h n iq u e o f B ondurant and
M i l l e r (1 9 6 2 ). Abrams in 1966 r e p o r te d f o r t h e f i r s t tim e t h e ;
'c h a r a c t e r i z a t i o n of a s u r f a c t a n t ag en t' w hich had been p r e v io u s ly
i !
i s o l a t e d from lu n g hom ogenates by means o f 'a new m ethod u t i l i z i n g |
a d i f f e r e n t i a l c e n t r i f u g a t i o n and s a l i n e g r a d i e n t te c h n iq u e
(Abrams and T a y lo r , 1 9 6 4 ). T h is l i p o p r o t e i n , i s o l a t e d in s a l i n e
i i
o f d e n s i t y 1 .1 5 showed a h ig h s u r f a c e a c t i v i t y and h ad a m o le c u la r
i
j . r
W eight c a l c u l a t e d from s u r f a c e t e n s i o n m easurem ents o f 2 . 4 i 0 . 5 x l 0 ^
I t s c h e m ic a l a n a l y s i s r e v e a le d 60 p e r c e n t p r o t e i n and 40 p e r cent-
i . i
f }
■Lipid. S e p a r a tio n of th e l i p i d s on s i l i c i c a c i d colum ns gave |
I ‘
(70% p h o s p h o lip id , 10% f a t t y a c i d , 14% t r i g l y c e r i d e , 6% f r e e |
! i
i i
c h o l e s t e r o l and l e s s th a n 0.5% c h o l e s t e r o l e s t e r . ;
; R e c e n tly , H a rla n , e t _al. (1 9 6 6 ), . s tu d y in g t h e pulm onary i
l i p i d c o m p o s itio n o f s p e c i e s w ith h ig h s u r f a c e a c t i v i t y (dog and
m a n ), and in s p e c i e s w ith lo w e r s u r f a c e a c t i v i t y ( f r o g , t u r t l e ,
and c h ic k e n ) , h av e found t h a t t h e t o t a l p h o s p h o lip id c o n t e n t o f
t h e lu n g was s i g n i f i c a n t l y h i g h e r in a n im a ls w i t h h ig h lu n g s u r - j
j i
| !
f a c e a c t i v i t y , an d t h a t q u a n t i t a t i v e d i f f e r e n c e s in p u lm o n ary j
I p h o s p h o lip id s w ere a s s o c i a t e d w ith d i f f e r e n c e s i n s u r f a c t a n t r e - j
!
l a t e d t o s p e c i e s .
: In t h e same y e a r , C l u t a r i o , e t _al. (1966) r e p o r t e d new
f i n d i n g s on th e c h e m ic a l c o m p o sitio n o f t h e s u r f a c t a n t a g e n t. j
S tu d y in g t h e f r a c t i o n s o b t a i n e d when lu n g e x t r a c t s w ere p a s s e d
th r o u g h g e l co lu m n s, th e y d e t e c t e d one f r a c t i o n c o n t a i n i n g l a r g e
m o le c u le s o r m i c e l l e s c o n t a i n i n g p h o s p h o lip id s an d p o ly s a c c h a r id e s ;
! I
jbut l i t t l e o r no p r o t e i n . T h is f r a c t i o n showed s u r f a c e a c t i v e j
p r o p e r t i e s .
i
So f a r , a l l t h e p a p e r s re v ie w e d h a v e f o c u s e d on t h e p h o s
p h o l i p i d f r a c t i o n , and m ore p r e c i s e l y on th e l e c i t h i n com pound, !
j |
a s t h e o n ly c h e m ic a l a g e n t r e s p o n s i b l e f o r s u r f a c e a c t i v i t y o f " j
t h e l i p o p r o t e i n com plex. N e v e r t h e l e s s , M cClenahan ( 1 9 6 7 ), working*
i . ;
jwith lu n g m a t e r i a l p u r i f i e d by .th e te c h n iq u e o f Abrams (1 9 6 6 ), hasj
l‘ i
i ' • «
d e m o n s tr a te d t h a t c y a n i d e - a l t e r e d s u r f a c t a n t h a s s i g n i f i c a n t l y j
5
j
d im in is h e d s u r f a c e a c t i v i t y . When th e c y a n i d e - a l t e r e d s u r f a c t a n t j
Was f r a c t i o n a t e d , t h e l i p i d com ponent d e m o n s tr a te d n o rm a l s u r f a c e
20
a c t i v i t y i n d i c a t i n g t h a t t h e p r o te in p o r tio n had been m o d ifie d
o r d e n a tu re d , b u t t h e p rim ary l i p i d component of t h e s u r f a c t a n t ,
d i p a l m i t o y l - l i c i t h i n , was u n a ffe c te d by p o ta ssiu m c y a n id e . A ccord
in g t o McClenahan (1 9 6 7 ), t h e s e s tu d i e s su g g e st t h a t th e s u r f a c e j
a c t i v i t y of th e s u r f a c t a n t can be s i g n i f i c a n t l y reduced by an
a l t e r a t i o n of i t s p r o te in m o iety a lo n e . At p r e s e n t, t h e r e f o r e , j
: !
one may co n clu d e t h a t t h e n a t i v e lu n g s u r f a c t a n t m a t e r i a l i s a i
com plex o f l i p i d s and p r o t e i n , and t h a t b o th a re e s s e n t i a l t o i t s :
b i o l o g i c a l a c t i v i t y .
Lung L in in g in V e r te b r a te s . . .
I . :
! The d is c o v e ry of th e s u rf a c e a c t i v e l i p o p r o t e i n in mammal- j
! I
\ \
Sian lu n g s prom oted a s e a rc h f o r i t in o th e r v e r t e b r a t e s . G ener- j
I s
I 1
a l l y sp e a k in g , one may say t h a t so f a r s u r f a c t a n t m a t e r i a l has
been found in a l l t h e mammals t h a t have been t e s t e d f o r i t : mouses,
jrat,' g u in e a p ig , r a b b i t , c a t , dog, sh e e p , cow, and human b e in g s ;
■ i
| !
;(C lem ents, 1962A ). !
I - |
In 1958 i t was r e p o r te d f o r th e f i r s t tim e ( P a t t l e , 1958)
[that f r o t h s from pig eo n and f r o g lungs behave in a s i m i l a r way as j
i :
i ■ ■ !
jthose from mammalian lu n g s . But in 1961, e x p e rim e n ts w ith s a l i n e j
1 ' ' j
E x tr a c ts o f am phibian, r e p t i l e , b i r d , and mammalian lu n g s , showed !
i !
p h a t t h e p re s e n c e o f a d i s t i n c t i v e s u r f a c e a c t i v e a g e n t was lim ited
21
t o mammalian lu n g s ( M i l l e r and B o n d u ra n t, 1 9 6 1 ). In o r d e r t o
r e c o n c i l e t h e s e c o n t r a d i c t o r y f i n d i n g s , P a t t l e and H opkinson
1(1963) re v ie w e d t h e p ro b le m . They c o n c lu d e d t h a t a s u r f a c e f ilm
c a p a b le o f r e d u c in g t o a l a r g e d e g re e t h e t e n s i o n o f t h e s u r -
I
1 . !
:f a c e s o f t h e f i n e a i r s p a c e s of t h e lu n g s e x i s t s in t h e b i r d , ■
j r e p t i l e , and a m p h ib ia n , a s w e ll a s in mammals. They a l s o found
: ' !
t h a t , a lth o u g h t h e b e h a v io r of b u b b le s from b i r d lu n g s was s im i- !
l i a r in a l l r e s p e c t s t o t h a t from mammals, t h e b e h a v io r of lu n g
b u b b le s from r e p t i l e s and a m p h ib ia n s was d i f f e r e n t : th e y w ere
r e l a t i v e l y u n s t a b l e . T h ese f i n d i n g s have b een i n t e r p r e t e d a s |
e v id e n c e t h a t t h e r e p t i l e and am phibian lu n g s have a l i n i n g n o t
i d e n t i c a l w ith t h a t o f b i r d o r mammal, b u t s t i l l c a p a b le o f j
lo w e r in g t h e s u r f a c e t e n s i o n ( P a t t l e , 1 9 6 5 ). i
I \
S in c e b i r d s , r e p t i l e s and a m p h ib ian s p r e s e n t s p e c i a l a n a
t o m i c a l and p h y s i o l o g i c a l f e a t u r e s d i f f e r e n t in some d e g re e from
. j
t h o s e o f mammals (H o ar, 1 9 6 6 ), t h e s e have been a n a ly z e d and i n t e r - j
! j
j 1
p r e t e d in such a way t h a t may l e a d t o a p o s s i b l e e x p l a n a t i o n o f j
t h e f u n c tio n o f t h e pulm onary s u r f a c e a c t i v e a g e n t i n lo w e r v e r te -j
i
i :
jb ra te s (C le m e n ts, 1962A) . A m phibians and r e p t i l e s , f o r exam ple,
t ;
j i
d ep en d more on t h e i r e n v iro n m e n t t o s u p p ly a m a jo r p o r ti o n o f t h e j
|
h e a t t h a t s u s t a i n s t h e i r m e ta b o lism ; w e ig h t f o r w e ig h t th e y do !
n o t r e q u i r e as much ex ch an g e o f r e s p i r a t o r y g a s e s as mammals, j
; i
i j
and t h e r e f o r e , do n o t n e e d as much lu n g s u r f a c e : t T h e i r - l u n g s . have^
22
l a r g e a i r sp a c e s b u t w ith l e s s s u r f a c e ; t h u s , th e y a r e n o t s e r i
o u s ly t h r e a t e n e d by t h e p r e s s u r e o f s u r f a c e t e n s io n c r e a te d by
t h e l i q u i d t h a t c o a ts t h e in n e r l i n i n g . I t seems r e a s o n a b le ,
t h e r e f o r e , t h a t r e p t i l e and am phibian lu n g s sh o u ld n o t r e q u i r e
'a h ig h ly e f f i c i e n t s u r f a c t a n t a g e n t as do mammalian lu n g s
fC le m e n ts, 1962A) . On t h e o th e r hand', b i r d s p o s s e s s lu n g s w ith j
a d d i t i o n a l a i r s p a c e s w hich a r e v e n t i l a t e d in a p e c u l i a r way. j
The r e s p i r a t o r y p ro c e s s i s a c co m p lish e d by draw ing a i r th ro u g h
\ i
t h e lu n g s i n t o l a r g e a i r sa c s c o n ta in in g th o u sa n d s o f a i r c a p i l - I
l a r i e s , in w hich gas exchange t a k e s p la c e and in t h i s way t h e
qhange o f volume in t h e a i r sp ace i s m in im ized . S in c e t h e r e i s j
I S
! 5
l i t t l e change in lu n g volum e, s u r f a c e t e n s io n does n o t need t o be
i
(ad ju sted (C le m e n ts, 1962A) . T h is f a c t , as s t a t e d by P a t t l e (1965),
I i
i ; i
m ig h t have l e d some i n v e s t i g a t o r s t o a c c e p t t h e a b se n c e of a s u r - i
f a c t a n t a g e n t in b i r d s ( M ille r and B o n d u ran t, 1 9 6 1 ). The co n -
| j
p l u s iv e f i n d i n g o f a s u r f a c t a n t in b i r d lu n g s by P a t t l e and j
! I
i • I
k o p k in so n (1963) s u g g e s ts t h a t t h e p rim a ry f u n c tio n o f th e s u r - |
f a c e a c t i v e a g e n t in b i r d s i s t o p re v e n t t r a n s u d a t i o n r a t h e r
; ■ - l
; |
th a n a l v e o l a r c o l l a p s e ( P a t t l e , 1 9 6 5 ). I
i ' I
! , j
j W ith r e s p e c t t o t h e o r ig i n o f t h e s u r f a c t a n t a g e n ts in |
! I
; ' !
b i r d s , r e p t i l e s and a m p h ib ia n s, i t h as been h y p o th e s iz e d by i
P a t t l e (1965) t h a t c h ic k e n s u r f a c t a n t h as a s i m i l a r o r ig i n t o I
i
i
I
23
t h a t in mammals, nam ely t h e ”a l v e o l a r c e l l s ” . In r e p t i l e s and in
a m p h ib ia n s, i t i s assum ed t h a t th e s u r f a c t a n t i s p ro d u ced by a
c l a s s o f c e l l s ( p r e v io u s ly d e s c r ib e d by B e r t a l a n f f y in 1 9 5 5 ),
id i s t i n c t * f r o m th o s e w hich form th e c o n tin u o u s v i s i b l e e p ith e liu m
■of t h e lu n g ( P a t t l e , 1965).
| I t i s im p o r ta n t t o m en tio n h e re t h a t a lth o u g h t h e p r e s e n c e
o f s u r f a c t a n t a g e n ts in v e r t e b r a t e s o th e r th a n mammals i s c l e a r l y
^ e s ta b lis h e d , t h e s e e x p e rim e n ts d id n o t p r o v id e e v id e n c e a s t o
iw hether such s u r f a c t a n t s have a s i m i l a r c o n s t i t u t i o n as t h a t of
mammals. T h e r e f o r e , much work rem ains t o be done in t h i s a r e a .
i In summary, I may say t h a t t h e f o llo w in g f in d i n g s have
|
been e s t a b l i s h e d :
jl. I t has been proven t h a t t h e lu n g a l v e o l i of mammals, b i r d s ,
* r e p t i l e s , and a m p h ib ia n s, p o s s e s s a l i n i n g f i l m w hich i s
h ig h l y i n s o l u b l e in w a te r and f r e e l y p e rm ea b le t o g a s e s .
;2. In mammals, t h e s u r f a c t a n t s t a b i l i z e s t h e dynam ic a c t i v i t y of
i
i
! t h e lu n g s . T h is i s a c c o m p lish e d by e q u a l i z i n g th e t e n s i o n in
t h e a i r s p a c e s and p e rh a p s by p r e v e n tin g o sm o tic f o r c e s from
!
d ra w in g f l u i d i n t o t h e a i r s p a c e . In b i r d s , i t a p p e a rs t h a t
I s u r f a c t a n t m ig h t a c t p r im a r i l y t o p r e v e n t t r a n s u d a t i o n .
i
:3. A l t e r e d s u r f a c e c h a r a c t e r i s t i c s o f t h e lu n g e x t r a c t s have
been fo u n d a s s o c i a t e d w ith s e v e r a l c l i n i c a l c o n d i t i o n s .
24
Among t h e s e a r e th e h y a lin e membrane d i s e a s e ( r e s p i r a t o r y
d i s t r e s s syndrome o f t h e n e w b o rn ), emphysema, pulm onary edema,
oxygen p o is o n in g , e x p o su re t o ch e m ica l i r r i t a n t s such as
to b a c c o smoke, o zo n e, smog, e t c e t e r a .
A r e l a t i o n s h i p has been shown betw een i n t r a u t e r i n e d e v e lo p
ment and c a p a b i l i t y t o form a l i n i n g f ilm by th e lu n g s . I n j
m ice, t h i s - c a p a b i l i t y a p p e a rs q u i t e su d d e n ly on t h e e i g h t e e n t h
day of em bryonic d ev elo p m en t when th e c o n tin u o u s l a y e r o f
c u b o id a l e p ith e liu m l i n i n g t h e lu n g s t a r t s t o d e sq u a m a te .
In human b e in g s , t h e a b i l i t y t o form a l i n i n g f il m d e v e lo p s
more g r a d u a lly d u r in g t h e f i f t h t o s e v e n th months o f g e s t a
t i o n , b u t in human f e t u s e s t h e a p p e a ra n c e of th e l i n i n g f i l m j
does n o t n e c e s s a r i l y c o in c id e w ith th e t o t a l d is a p p e a ra n c e
of t h e lu n g c u b o id a l e p ith e liu m .
The o r i g i n o f t h e s u r f a c t a n t h a s been, s tu d i e d in some d e t a i l
and much i s known a b o u t th e s i t e o f s y n th e s is of some p h o s
p h o lip id s in mammals, b u t n o th in g w ith r e g a rd t o t h e s i t e o f |
s y n th e s is o f t h e p e p t i d e m o ie ty , and o f t h e s i t e o f b in d in g j
o f l i p i d s w ith p e p t i d e s t o form t h e whole b i o l o g i c a l l y a c t i v e j
l i p o p r o t e i n .
The mammalian- s u r f a c e a c t i v e m a t e r i a l h a s been i d e n t i f i e d as !
a l i p o p r o t e i n com plex, and i t s l i p i d co m p o sitio n h a s been
25
f a i r l y w e ll e s t a b l i s h e d , b u t n o th in g i s known ab o u t t h e com
p o s i t i o n and s t r u c t u r e of i t s p e p tid e m o ie ty , and v i r t u a l l y
n o th in g i s known a b o u t th e s p a t i a l r e l a t i o n s h i p s betw een t h e
l i p i d and p e p tid e m o i e t i e s .
7 . A lth o u g h i t h as been b e lie v e d t h a t t h e s u r f a c e a c t i v i t y p r o
p e r t y o f t h e s u r f a c t a n t a r i s e s from i t s p h o s p h o lip id compon
e n t s , to d a y t h e r e e x i s t s e x p e r im e n ta l e v id e n c e t h a t t h e w hole
n a t i v e lu n g l i p o p r o t e i n i s r e s p o n s i b le f o r t h i s p r o p e r ty
r a t h e r th a n t h e p h o s p h o lip id s a l o n e .
!'8. I t i s o b v io u s t h a t a t p r e s e n t t h e r e a r e a number o f problem s
r e l a t e d t o t h e s u r f a c e a c t i v e l i p o p r o t e i n w hich rem ain t o be
i n v e s t i g a t e d . Among them t h e r e i s one whose s o l u t i o n w i l l
b r i n g fu n d a m e n ta l in fo rm a tio n n e c e s s a r y f o r th e d e s ig n of
new e x p e r im e n ts . In my o p in io n , a b e t t e r know ledge o f th e
c h e m ic a l and p h y s ic o c h e m ic a l c h a r a c t e r i s t i c s o f t h e p e p tid e
j m o ie ty of t h e l i p o p r o t e i n complex i s such a p ro b le m .
S ta te m e n t o f th e P ro b lem
; The p r e s e n t stu d y i s co n cern ed w ith e s ta b li s h m e n t o f t h e
j
jc h em ical and some of t h e p h y s ic o c h e m ic a l c h a r a c t e r i s t i c s of t h e
ip e p tid e m o ie ty o f t h e s u r f a c e a c t i v e l i p o p r o t e i n i s o l a t e d a c c o r d
i n g t o t h e abram s te c h n iq u e (Abrams, 1 9 6 6 ). I t has been
u n d e r ta k e n in t h e hope t h a t new f i n d i n g s may e x te n d p r e s e n t know
le d g e o f t h e s t r u c t u r e o f t h e s u r f a c e a c t i v e a g e n t and o f l i p o
p r o t e i n s in g e n e r a l .
P r e c i s e i n f o r m a tio n c o n c e r n in g t h e c h e m ic a l c o m p o s itio n o f
|th e p e p t i d e m o ie ty , p a r t i c u l a r l y i t s am ino a c i d c o m p o sitio n and
ith e p a r t i c i p a t i o n o f c a r b o h y d r a te s in i t s c o m p o s itio n w i l l be
p r e s e n t e d . U l t r a c e n t r i f u g a l s t u d i e s o f t h e p e p t i d e m o ie ty w i l l
ia ls o be r e p o r t e d .
F i n a l l y , I w ould l i k e t o m e n tio n t h a t i t i s th o u g h t t h a t ,
:by know ing more a b o u t t h e n a t u r e o f lu n g s u r f a c t a n t , i t w ould be
p o s s i b l e in f u t u r e i n v e s t i g a t i o n s t o u n d e r s ta n d b e t t e r t h e p a r t i -
i
I
!
i c i p a t i o n o f t h i s l i p o p r o t e i n in t h e p a th o lo g y o f t h e r e s p i r a t o r y
^ d i s t r e s s syndrom e o f t h e n ew b o rn , w h ere t h e r e may be a r e l a t i o n
s h i p b etw een d e f e c t i v e i n h e r i t e d b i o s y n t h e s i s o f t h e l i p o p r o t e i n
jcomplex and t h e d i s e a s e . O r, i t w ould b e p o s s i b l e t o d e s ig n new
je x p e rim e n ts f o r s tu d y i n g t h e o r i g i n o f t h e l i n i n g com plex, o r i t s
i
.tr a n s f o r m a tio n i n t o t h e i n s o l u b l e l i n i n g f i l m , u t i l i z i n g m odern
t e c h n i q u e s w ith l a b e l e d am ino a c i d s , o r s p e c i f i c f l u o r e s c e n t o r
lla b e le d a n t i b o d i e s .
27
Acknowledgem ents
I am m ost g r a t e f u l t o D r. Samuel E . A l l e r t o n , member of my
G u id an ce C om m ittee, f o r h i s o r i g i n a l s u g g e s tio n of t h e p ro b le m , i
c o n tin u e d a d v ic e and en co u rag em en t, f o r te a c h i n g me many u s e f u l
jla b o r a to r y t e c h n i q u e s , and f o r re a d in g c r i t i c a l l y t h e f i r s t d r a f t j
o f t h i s D i s s e r t a t i o n . A p p r e c ia tiv e th a n k s a r e due t o D r. P a u l R. i
■ S aunders (C h airm a n ), D r. J a y M. Savage, D r. A rnold Dunn, and Dr,
R o b e rt M. Chew, who s e rv e d on t h e G uidance Committee and c r i t i -
icized t h e m a n u s c rip t.
The f o llo w in g i n d i v i d u a l s k in d ly s u p p lie d specim ens f o r
! f
jthe p r e s e n t s tu d y : D r. W illia m M. Blackm ore ( D i r e c t o r o f t h e j
jVivarium of t h e U n iv e r s ity of S o u th ern C a l i f o r n i a ) , D r. M urray B. j
i !
' i
G a rd n e r (C o m p arativ e P a t h o l o g i s t , Los A n g eles C o u n ty - U n iv e r s ity ,
o f S o u th e rn C a l i f o r n i a M e d ic a l C e n te r ) , and Mr. Ben L i l i e n (Los ,
A n g e le s Meat Company, V ernon, C a l i f o r n i a ) . To a l l I e x te n d my
I ■ !
I I
jsin c e re . a p p r e c i a t i o n . I a l s o want t o th a n k D r. L u c ie n A. B a v e tta ,j
j ^ I
Chairm an of t h e D epartm ent o f B io c h e m is try , S ch o o l o f D e n t i s t r y , :
{for p e r m it t i n g t h e use o f equipm ent o f h i s l a b o r a t o r y d u r in g t h i s I
i n v e s t i g a t i o n . j
I !
The a u th o r w ish es t o e x te n d s p e c i a l th a n k s to D r. Myles j
M a x fie ld and D r. J a y M. S avage, f o r g u id a n c e , e n co u rag em en t, and i
j
many p e r s o n a l k in d n e s s e s d u r in g h i s g r a d u a te s t u d i e s . ________________
28
Economic s u p p o rt d u rin g t h e s e s tu d ie s h a s been p ro v id e d by
a f e llo w s h ip from th e U n iv e r s ity of C o sta R ic a , and a r e s e a r c h
a s s i s t a n t s h i p from th e A llan Hancock F o u n d a tio n , U n iv e r s ity of
S o u th ern C a l i f o r n i a . I w ish t o acknow ledge, f i n a l l y , th e lo n g -
s u s ta in e d encouragem ent and su p p o rt o f D r. L e s l i e A. Chambers,
iD ire c to r of th e A lla n Hancock F o u n d a tio n .
CHAPTER I I
MATERIALS AND METHODS ;
I s o l a t i o n an d c h a r a c t e r i z a t i o n o f t h e lu n g s u r f a c e - a c t i v e
l i p o p r o t e i n c o m p le x . '
i
O r d in a r y l a b o r a t o r y c h e m ic a ls u s e d i n t h i s r e s e a r c h
w ere o f a n a l y t i c a l r e a g e n t g r a d e . U n le s s s t a t e d o t h e r w i s e ,
o p t i c a l d e n s i t i e s f o r e a c h t e s t were r e a d w ith 1 cm q u a r t z
c u v e t t e s in t h e Beckman DB S p e c tr o p h o to m e te r (Beckman I n s t r u - j
m e n ts I n c o r p o r a t e d , P a l o A l t o , C a l i f o r n i a ) .
D u rin g t h e e x p e r i m e n t a l p a r t o f t h i s s t u d y , e i g h t l o t s j
o f t h e lu n g s u r f a c e - a c t i v e l i p o p r o t e i n com plex ( c a l l e d SA-
l i p o p r o t e i n th r o u g h o u t t h e t e x t ) w ere i s o l a t e d fro m f r e s h lu n g
t i s s u e s : tw o l o t s fro m g u in e a p ig s ( H a r t l e y s t r a i n fro m t h e I :
V iv a riu m o f t h e U n i v e r s i t y o f S o u th e rn C a l i f o r n i a ) ; t h r e e I
l o t s from human b e in g s ( o b t a i n e d p o s t-m o rte m fro m t h e Los
A n g e le s C o u n t y - U n i v e r s i t y o f S o u th e rn C a l i f o r n i a M e d ic a l ;
C e n t e r ) ; an d t h r e e l o t s from cows (fro m t h e Los A n g e le s M eat !
|
Company, V e rn o n , C a l i f o r n i a ) . I
30
A . I s o l a t i o n ;
S A - lip o p r o te in s w ere i s o l a t e d from t h e lu n g s a c c o r d in g
t o t h e p ro c e d u re of Abrams (1 9 6 6 ), as f o llo w s : f r e s h lu n g
t i s s u e was hom ogenized w ith 0 ,8 5 p e r c e n t sodium c h l o r i d e I
i
( M a llin c k r o d t) s o l u t i o n , c o n ta in in g 0 .0 1 p e r c e n t o f sodium '
s a l t o f EDTA ( E t h y l e n e d i n i t r i l o ) t e t r a a c e t i c a c id d iso d iu m '
s a l t , (Eastm an O rg a n ic C h e m ic a ls), 5 m l/g o f lu n g , u s in g a i
S e r v a l l Omnimixer (Iv a n S o r v a l l , I n c o r p o r a te d , N orw alk,
C o n n e c tic u t) w ith 450 m l s t a i n l e s s s t e e l c u p s, f o r f i v e m in
u t e s a t 16,000 r .p .m . The hom ogenate was c h i l l e d in an i c e
b a th . T h is hom ogenate was c e n t r i f u g e d a t 1,500 r .p .m . (300. g)|
f o r te n m in u te s a t + 4 °, u s in g a S e r v a l l r e f r i g e r a t e d - a u t o m a t i c
j
c e n t r i f u g e (Iv a n S o r v a l l , I n c o r p o r a te d , N orw alk, C o n n e c tic u t) j
w ith an 5 S -3 4 r o t o r (4 .2 5 in c h e s , r a d i u s ) . The s u p e r n a t a n t ,
o r " s a l i n e e x t r a c t " was th e n a s p i r a t e d and c e n t r i f u g e d f o r
one h o u r a t 3,000 r .p .m . (1 ,0 0 0 g ) ' a t + 4 °. The s u p e r n a t a n t j
was d i s c a r d e d and t h e p r e c i p i t a t e su sp en d ed in 1 .1 5 0 s p e c i f i c I
j
g r a v i t y sodium c h l o r id e s o lu t i o n and c e n t r i f u g e d a t 3 ,7 5 0 ;
r .p .m . (1 ,5 0 0 g) f o r t w e n t y - f i v e m in u te s a t + 4 °, u s in g t h e |
same t y p e o f r o t o r and c e n t r i f u g e . The c o n te n ts o f th e tu b e s
s e p a r a t e i n t o t h r e e f r a c t i o n s : an upperm ost p e l l i c l e , an un-
se d im e n te d f r a c t i o n , and a p e l l e t a t t h e b o tto m o f th e t u b e s , j
31
The p e l l i c l e c o n t a i n i n g s u r f a c e - a c t i v e l i p o p r o t e i n was a s p i r
a t e d and th e n d i a l y z e d a g a i n s t 0.85% sodium c h l o r i d e c o n t a i n
i n g 0 .0 1 p e r c e n t o f t h e sodium s a l t of EDTA. The f l o t a t i o n
and p e l l e t i n g o f t h e l i p o p r o t e i n w ere r e p e a t e d t w i c e . The j
j
S A - l ip o p r o t e i n c o n c e n t r a t i o n s in t h e s e d i a l y s a t e s w ere e s t i - !
m ated by d r y in g a l i q u o t s t o c o n s t a n t w e ig h t. I
I
B . L i p o p r o t e i n c o n t e n t on t h e s a l i n e d i a l y z e d e x t r a c t s |
The d r y w e ig h t o f t h e m a t e r i a l t o b e used i n t h e f o ll o w
i n g p h y s i c a l and c h e m ic a l e x p e r im e n ts was d e te rm in e d by p l a c - ;
i n g t h e l y o p h i l i z e d , i s o l a t e d S A - l ip o p r o t e i n m a t e r i a l s i n t o
a w e ig h in g b o t t l e o f known d ry w e ig h t. The m a t e r i a l was j
i
d r i e d a t 1 1 0 ° o v e r p h o sp h o ru s p e n to x id e (M ath eso n ) in a d e s s i- i
c a t o r . The m a t e r i a l was w eig h ed b e f o r e d r y in g was b eg u n , and j
■ |
a t t w e n t y - f o u r h o u r i n t e r v a l s u n t i l a c o n s ta n t w e ig h t was
o b t a i n e d . The d i f f e r e n c e b etw een t h e w e ig h t b e f o r e and a f t e r >
d r y in g was r e c o r d e d a s t h e m o is tu r e c o n t e n t . I n o u r e x p e r i
m e n ts , tw o w e ig h in g b o t t l e s w ere d r i e d a t 110° o v e r p h o sp h o ru s
p e n to x id e i n a d e s s i c a t o r a t vacuum f o r f o u r t y - e i g h t h o u r s .
B e fo re w e ig h in g , d e s s i c a t o r and b o t t l e s w ere a llo w e d t o r e a c h
room t e m p e r a t u r e . Then t h e c a p p e d b o t t l e s w ere a llo w e d t o
o u t s i d e e q u i l i b r a t e w ith a i r i n s i d e t h e c l o s e d b a la n c e i
!
( M e t t l e r I n s t r u m e n t s C o r p o r a t io n , H ig h ts to w n , New J e r s e y ) f o r I
t h i r t y m in u te s . T h is' was done a t tw e n ty - f o u r h o u r i n t e r v a l s . :
A liq u o ts o f 50.0 m l, m easu red w ith v o lu m e tric p i p p e t e s , of
t h e m a t e r i a l s under s tu d y , w ere l y o p h i l i z e d and t h e i r con
s t a n t w e ig h t was d e te r m in e d . C o r r e c tio n s w ere made f o r th e
c o n t r i b u t i o n s of sodium chloride-E D TA s a l t s .
The p r o te in c o n t e n t o f d r i e d S A - lip o p r o te in was e s t i - j
i
m ated on t h e b ases o f i t s n i t r o g e n c o n te n t. j
C . C h em ical c h a r a c t e r i z a t i o n o f t h e S A - lip o p r o te in
In o r d e r t o e s t a b l i s h t h e ch e m ic a l co m p o sitio n of th e
m a t e r i a l s i s o l a t e d in o u r l a b o r a t o r y , a s e r i e s o f t e s t s were
• c a r r i e d o u t .
i
1 . D e te rm in a tio n o f p r o t e i n c o n te n t j
|
j
a . T o ta l N i t r o g e n . T ech n ico n M ic ro -K je ld a h l (1 9 6 5 ). j
I
The q u a n t i t a t i v e d e te r m in a tio n o f t o t a l n itr o g e n
in t h i s m ethod in v o lv e s co m p le te d i g e s t i o n o f o r g a n ic m a te r
i a l in a c o n tin u o u s d i g e s t o r u n i t w ith a d i g e s t i o n m ix tu re
c o n t a i n i n g 3 grams o f S elen iu m D io x id e (M atheson Coleman &
B e ll) d i s s o lv e d in 100 m l o f d i s t i l l e d w a te r added slo w ly t o I
900 ml o f c o n c e n tr a te d s u l f u r i c a c id (B ak er & A dam son), and j
fo llo w e d by th e a d d i t i o n o f 20 ml o f 70 p e r c e n t p e r c h l o r i c I
|
a c id ( M a llin c k r o d t. D e te r m in a tio n o f t h e ammonia in t h e j
r e s u l t i n g d i g e s t i s a c h ie v e d w ith a p h e n o l- h y p o c h lo r ite s y s - j
tern, by w hich ammonia i s c o n v e rte d t o a b lu e c o lo re d compound
33
o f t h e in d o p h e n o l t y p e .
Sam ples and d i g e s t i o n m ix tu re w ere p r o p o r tio n e d , and
f e d i n t o t h e d i g e s t o r u n i t a u t o m a t i c a l l y . A f t e r d i g e s t i o n ,
d i l u e n t w a te r was added and a f t e r m ix in g , a p o r tio n o f t h e i
d i l u t e d d i g e s t was c o n tin u o u s ly a s p i r a t e d i n t o t h e c o l o r i
m e t r i c sy ste m . I t was f i r s t n e u t r a l i z e d w ith 35 p e r c e n t
sodium hyd ro x y d e ( M a llin c k r o d t) s o l u t i o n , th e n a l k a l i n e phenol!
( p r e p a r e d by d i s s o l v i n g 272 m l o f l i q u i f i e d 88 p e r c e n t p h e n o l
( M a llin c k r o d t) in 500 ml o f 40 p e r c e n t sodium hyd ro x y d e s o l u
t i o n ) , and 5 p e r c e n t sodium h y p o c h l o r it e ( M a llin c k r o d t) w ere i
i
s u c c e s s i v e l y a d d ed , m ix in g t a k i n g p la c e betw een r e a g e n t a d d i-
| t i o n s . A f t e r p a s s i n g th ro u g h a tim e d e la y c o i l , th e r e s u l t i n g
i c o l o r was m easured a t 630 mp,. S ta n d a r d s o f ammonium s u l f a t e
i !
( M a llin c k r o d t) r a n g in g from 50(-ig./ml t o 1 , OOOug/ml w ere run
s im u lta n e o u s ly and u s e d f o r c o m p a riso n . A f a c t o r o f 6.25 was ;
u sed f o r c a l c u l a t i o n o f p r o t e i n c o n te n t from t o t a l n i t r o g e n ;
| • j
c o n t e n t . j
2 . L ip id a n a l y s i s
a . L ip id e x t r a c t i o n
I .
l j
! The l i p i d c o m p o sitio n o f S A - lip o p r o te in was j
! !
i I
i .
I s tu d i e d u s in g l i p i d e x t r a c t s o b ta in e d a t v e ry low t e m p e r a tu r e j
i
! w ith 2 :1 c h lo ro fo rm -m e th a n o l ( v /v ) by f r e e z i n g and th a w in g ;
I i
i I
t h r e e t i m e s . One m l o f t h e l i p o p r o t e i n s u sp e n s io n was added
34
dro p w ise w ith s t i r r i n g in a g ra d u a te 15 ml c e n t r i f u g e tu b e ,
t o 9' ml of 2 :1 c h lo ro fo rm -m e th a n o l ( M a llin c k r o d t) . Tubes
were capped, i n v e r te d f o r m ix in g and immersed in a 'm ix tu r e
of 2 -e th o x y e th a n o l (M atheson) and d ry i c e , u n t i l f r o z e n . The j
i
s o lu tio n was allo w ed t o thaw and ag ain f r o z e n . T h is p ro c e ss
i
was r e p e a te d t h r e e t i m e s . Then th e m ix tu re was c e n tr if u g e d ;
j
a t 6,000 r .p .m . f o r te n m in u tes in a S e r v a l l r e f r i g e r a t e d -
a u to m a tic c e n t r i f u g e . The u pper aqueous l a y e r was removed by :
th e u s e of a P a s te u r p ip p e te and d is c a r d e d . Enough c h lo r o -
form -m ethanol m ix tu re was added t o th e tu b e s in o r d e r t o i
b r in g th e f i n a l volume of- t h e o rg a n ic p h a se t o 10 m l. Tubes
were in v e r te d f o r m ixing and f o u r 2 . 0 m l sam p les, f o r d u p l i - j
c a te c h o l e s t e r o l and phosphorous a n a l y s i s , were p u t in t e s t j
j
t u b e s . The c h lo ro fo rm -m e th a n o l s o lv e n ts were removed by
h e a tin g th e t e s t tu b e s on a h o t p l a t e a t 80° u n t i l j u s t d ry .
b. P h o s p h o lip id s j
‘
P hosphorus was e s tim a te d a c c o rd in g t o F is k e and
Subarrow (1 9 2 5 ). F o r t o t a l p h o sp h o ru s, th e sam ples were j
d ig e s te d a t 160° in a p e r c h l o r i c - s u l f u r i c a c id m ix tu re u n t i l j
i
th e s o lu tio n was c l e a r (Lowry, e t a l . , 1 954). The c o lo r was
s u b se q u e n tly d e v e lo p e d by t h e p ro c e d u re o f B a r t l e t t (1 9 5 9 ).
P h o s p h o lip id s were c a l c u l a t e d by m u ltip ly in g l i p i d p hosphorus j
35
v a lu e s by 2 5 (a ssu m in g 4 p e r c e n t o f t o t a l p h o sp h o ru s p e r m ol
o f l e c i t h i n ) . To e a c h t u b e c o n t a i n i n g d r i e d l i p i d e x t r a c t
fro m t h e S A - lip o p r o te in 0 .6 m l o f an T ,a s h in g m i x t u r e ” was
a d d ed ( p r e p a r e d by a d d in g 1 6 .2 5 m l 70 p e r c e n t o f p e r c h l o r i c j
j
a c i d (M atheson) and 70 ml o f c o n c e n t r a t e d s u l f u r i c a c id
(B a k e r & Adamson) t o d i s t i l l e d w a t e r in a v o lu m e tr ic f l a s k |
and d i l u t e d t o t h e m ark o f 250 m l ) . S o l u t i o n s w ere a llo w e d
t o r e a c h room t e m p e r a tu r e an d 5 .0 m l o f 0 .2 p e r c e n t ammonium .
m o ly b d a te was added t o e a c h t u b e . Tubes w e re p u t i n a b o i l i n g
w a te r b a th f o r te n m in u te s and t h e n c o o le d t o room te m p e ra tu re '.
The c o lo r d e v e lo p e d was r e a d in a K le tt-S u m m e rso n p h o t o e l e c
t r i c c o l o r im e t e r ( K l e t t MFG Company, New Y o rk ) u t i l i z i n g t h e j
' !
No. 6 6 f i l t e r (620 t o 680m(ji) . S ta n d a r d s o f p h o sp h o ru s j
i
(N a2 HPC>4 • 7 H2 O d i s s o l v e d i n d i s t i l l e d w a te r ) r a n g in g from
1.0p.gP/m l t o 20.0|_igP/ml w e re run s i m u l t a n e o u s l y and used f o r
c o m p a riso n . A r e a g e n t b la n k was' a l s o run e a c h t i m e . I
f
j
c . T o t a l c h o l e s t e r o l
The m ethod u s e d was t h a t recommended by S e a rc y
and B e r g q u is t ( 1 9 6 0 ). An o ra n g e c o l o r i s form ed when ch o - j
i
j
l e s t e r o l d i s s o l v e d i n an a c e t o n e - e t h a n o l m ix tu r e and a s o l u - j
I
t i o n of FeSO^ in g l a c i a l a c e t i c a c i d a r e t r e a t e d w i t h co n - j
c e n t r a t e d H2 SO4 . T h is c o l o r can b e q u a n t i t a t e d a t a j
w a v e le n g th l e s s s u s c e p t i b l e t o i n t e r f e r i n g s u b s t a n c e s (470
t o 530m|t) . O n e - h a lf m l o f 1 : 1 a c e t o n e - e t h a n o l ( v / v ) was
ad d ed t o e a c h o f th e . t u b e s c o n t a i n i n g t h e d r i e d l i p i d e x t r a c t j
o f t h e S A - l i p o p r o t e i n and m ix e d , th e n 6 .0 ml o f F e S O ^ - a c e tic
a c i d r e a g e n t (A s a t u r a t e d s o l u t i o n o f FeSO^ ( B a k e r ’s ) i n
g l a c i a l a c e t i c a c i d (B a k e r & Adamson ) ) was a d d e d , and s t i r r e d ;
5
Two m l o f c o n c e n t r a t e d s u l f u r i c a c i d (B a k e r & Adamson) was I
blow n i n t o e a c h t u b e and m ix ed in r a p i d l y w ith an a u to m a tic j
I
m ix e r . T ubes w ere a llo w e d t o c o o l t o room t e m p e r a t u r e an d
t h e d e v e lo p e d c o l o r was r e a d i n a K le tt-S u m m e rso n p h o to
e l e c t r i c c o l o r i m e t e r ( K l e t t , MFG Company, New Y o r k ) , u s in g
t h e No.. 50 f i l t e r (470 t o 530 m|i) . C h o l e s t e r o l s t a n d a r d s
i
( p u r i f i e d c h o l e s t e r o l from N u t r i t i o n a l B io c h e m ., d i s s o l v e d j
j
i n c h lo r o f o r m ) r a n g in g from 40p,g/ml t o 2 0 0 |ig /m l w ere s im u l - j
t a n e o u s l y ru n an d u s e d f o r c o m p a ris o n . S am ples w ere r e a d
a g a i n s t - a b la n k o f r e a g e n t s ,
d . T r i g l y c e r i d e s
T r i g l y c e r i d e s w e re d e te r m in e d a c c o r d i n g t o t h e 1
\
1
m ethod o f Van H a n d e l an d Z i l v e r s m i t ( 1 9 6 7 ) . T h is m ethod !
i s b a s e d upon m e a s u rin g t h e g l y c e r o l m o ie ty o f t r i g l y c e r i d e s .
j
The e x t r a c t i o n o f l i p i d s fro m sam p le s i s a c c o m p lis h e d w i t h j
t h e a i d o f a z e o l i t e ( D o u c il) and c h lo r o f o r m ( F o r b e s , 1 9 3 0 ),
and t h e i r g l y c e r o l c e n t e n t i s d e te r m in e d e s s e n t i a l l y a c c o r d i n g
t o Lamber and N eish (1 9 5 0 ).
F our grams of a c t i v a t e d D o u c il1 (T . A. T a y lo r Company,
B a ltim o re , M aryland, a c t i v a t e d by h e a t i n g f o r f o u r h o u rs in
an oven a t 125°) was p u t in a g la s s - s to p p e r e d tu b e and 2 ml
o f c h lo ro fo rm was added and -shaken. One ml of t h e SA-
l i p o p r o t e i n su sp e n sio n was p u t on t h e to p o f t h e D o u c il,
mixed th o ro u g h ly and 18 ml o f ch lo ro fo rm was added. The
s to p p e re d tu b e s were shaken i n t e r m i t t e n t l y b u t v ig o ro u s ly f o r
te n m in u te s, and th e s o lu tio n f i l t e r e d th ro u g h a c o a rse f a t -
f r e e f i l t e r p a p e r. T r i p l i c a t e a l i q u o t s o f 2 .0 ml of t h e
c l e a r f i l t r a t e were m easured i n t o t h r e e g l a s s t u b e s . At
t h i s p o i n t , t h r e e 1 . 0 ml p o r tio n s o f a t r i g l y c e r i d e s ta n d a rd
s o lu tio n ( 0 . 0 5mg/ml, p re p a re d by d i s s o l v i n g 0.50 gm o f com
m e r c ia l corn o i l in c h lo ro fo rm t o 1 0 0 ml v o lum e), were mea
s u re d i n t o t h r e e g l a s s t u b e s . The s o lv e n t from a l l tu b e s
was e v a p o ra te d by p u t t i n g t h e tu b e s on a h o t p l a t e a t 80 °.
To two o u t o f each t h r e e s ta n d a rd s and unknowns, 0 .5 0 ml of
a l c o h o l i c KOH was added (p re p a re d by d i s s o l v i n g 2 gm of KOH
in 95 p e r c e n t r e d i s t i l l e d e t h y l a lc o h o l and d i l u t i n g t o 100
m l; b e f o re u s e , 1 0 ml o f t h i s s to c k s o lu t i o n was d i l u t e d t o
-'-Doucil i s a s y n t h e t i c z e o l i t e (sodium s i l i c o a lu m in a te )
38
50 ml w ith 95 p e r c e n t a lc o h o l) ( S a p o n if ie d s a m p le s ) . To t h e :
t h i r d tu b e s 0 .5 0 ml o f 95 p e r c e n t a lc o h o l was added (U nsap
o n i f i e d s a m p le s ) . A l l t h e tu b e s were k e p t a t 60° t o 70° f o r ;
f i f t e e n m in u tes on a h o t p l a t e . Then 0 .5 0 ml o f a 0 .2 N H2 SO4]
s o lu t i o n was added and th e tu b e s were p la c e d in a b o i l i n g
w a te r b a th f o r f i f t e e n m in u te s t o remove t h e a l c o h o l . A f te r j
1
c o o lin g th e g l y c e r o l c o n te n t o f t h e sam ples was d e te rm in e d j
by a d d in g 0 .1 0 ml o f a 0 .0 5 M p e r i o d a t e s o lu t i o n (1 .0 7 gm o f
NalO^ (M atheson Coleman £ - B e l l ) d i s s o l v e d in 100 ml o f d i s
t i l l e d w a te r) t o each tu b e and a f t e r te n m in u te s t h e o x id a tio n ;
p r o c e s s was sto p p e d by a d d i t i o n of 0 .1 0 ml o f a 0 .5 M sodium . :
a r s e n i t e r e a g e n t (p re p a re d by d i s s o l v i n g 2 .2 5 gm o f NaOH and
5 .0 gm o f a r s e n i c t r i o x i d e (B ak er C hem ical Company) in d i s -
i
j
t i l l e d w a te r t o 100 ml v o lu m e). A f t e r m ix in g , t o t h e d e a r ;
s o lu t i o n o f th e t u b e s , 5 .0 ml o f a c h ro m o tro p ic a c id r e a g e n t ;
(p re p a re d by d i s s o l v i n g 2 .0 gm 4 ,5 d ih y d r o x y - 2 . 7 - n a p h th a le n d i- |
' j
s u l f o n i c a c id (M atheson Coleman & B e l l ) in 200 ml o f d i s t i l l e d j
w a te r, and a d d in g 600 ml o f c o n c e n tr a te d r e a g e n t s u l f u r i c
a c id d i l u t e d w ith 300 ml o f d i s t i l l e d w a t e r ) , was added w ith I
m ix in g and th e tu b e s were h e a te d f o r o n e - h a lf h o u r a t 1 0 0 ° in
a b o i l i n g w a te r b a th in t h e ab sen ce o f d i r e c t l i g h t . A f te r
c o o lin g t o room te m p e r a tu re t h e o p t i c a l d e n s i t i e s o f th e
39
s o l u t i o n s w ere re a d a t 570mu in t h e Beckman DB S p e c tr o p h o to
m e t e r . R e s u l t s w ere c a l c u l a t e d by t h e f o ll o w i n g f o rm u la :
(O .D . s a p o n i f i e d unknow n)- ( 0 .D . u n s a p o n if ie d unknown) x q_5 q x
V J 7 J T . s a p o n i t i e d s t a n d a r d ; - (O .D . s a p o n i f i e d s t a n d a r d ; !
2 ? 0 0 0 _____ = rag p e r c e n t t r i g l y c e r i d e s j
ml c h lo r o f o r m e x t r a c t I
I
e . F a t t y a c i d s j
The m ethod p ro p o s e d by S t e r n and S h a p ir o (19 5 3 ) j
was u t i l i z e d h e r e . H ig h e r f a t t y a c i d e s t e r s r e a c t a t room ,
t e m p e r a t u r e w ith h y d ro x y la m in e in a l k a l i n e s o l u t i o n s i n a q u e - j
ous a l c o h o l . The p r e s e n c e o f a l c o h o l makes t h e r e a c t i o n p o s - :
s i b l e and a l s o k e e p s t h e lo n g - c h a in h y d ro x a m ic a c id s in s o l u - j
t i o n . T h e se a c i d s p ro d u c e a r e d - t o - v i o l e t c o l o r w ith f e r r i c |
j
c h l o r i d e w h ic h makes p o s s i b l e t h e i r q u a n t i t a t i v e e s t i m a t i o n \
( S te r n and S h a p i r o , 1 9 5 3 ). j
One ml o f t h e S A - l ip o p r o t e i n s u s p e n s io n was d e l i v e r e d
i n t o a g r a d u a t e 15 m l c e n t r i f u g e t u b e c o n t a i n i n g a p p r o x im a te ly
8 ml o f a 3 :1 a l c o h o l ^ e t h e r m ix tu re ( t h r e e p a r t s o f a b s o l u t e
e t h y l a l c o h o l and one p a r t o f d i e t h y l e t h e r ) . T h is m ix tu r e !
I
i
i
was b ro u g h t t o b o i l i n g , c o o le d , c e n t r i f u g e d , an d t h e u p p e r \
j
a q u e o u s ■ l a y e r was d i s c a r d e d . The f i n a l volum e o f t h e o r g a n ic j
N |
m i x tu r e was made up t o 1 0 .0 ml by a d d in g 3 :1 a l c o h o l - e t h e r |
!
( v / v ) . A f t e r i n v e r t i n g f o r m ix in g , 3 .0 ml ( i n d u p l i c a t e ) j
a l i q u o t s o f t h e m i x tu r e w ere d e l i v e r e d i n t o t e s t t u b e s . A
40
b la n k c o n ta in in g 3 .0 ml o f th e a l c o h o l - e t h e r s o lu t i o n was
in c lu d e d in e v e ry r u n . O n e -h a lf ml of a 2 M hyd ro x y lam in e
h y d r o c h lo rid e r e a g e n t (p re p a re d by d is s o l v i n g ' 13.89 cm of
NH^OH.Cl ( B a k e r’ s) in d i s t i l l e d w a te r in a 100 ml v o lu m e tric
f l a s k ) , and 0 .5 0 ml of 3 .5 N sodium hydroxyde s o lu t i o n were
ad d ed . The s to p p e re d tu b e s were s t i r r e d and a llo w ed to * s ta n d
f o r tw e n ty m in u tes a t room te m p e r a tu r e . Then 0 .6 0 ml o f a
h y d r o c h lo r ic s o lu t i o n (one volume of c o n c e n tr a te d HC1 d i l u t e d
w ith two volumes o f d i s t i l l e d w a te r) was added, and f i n a l l y ,
0 .5 0 ml of a 0 .3 7 M - f e r r i c c h l o r id e r e a g e n t (p re p a r e d by d i s
s o lv in g 3.42 gm o f F eC l2 (B aker -& Adamson) in 0 .1 N HC1 t o
1 0 0 ml volume) was added and t h e tu b e s were th o ro u g h ly m ixed.
O p t i c a l d e n s i t i e s of th e s o l u t i o n s w ere re a d a t 525mu in th e
Beckman DB S p e c tro p h o to m e te r. S ta n d a rd s o f t h e f a t t y a c id
t r i a c e t i n (E a stm a n ), d i s s o lv e d in 3 :1 a l c o h o l - e t h e r s o lu t i o n
and ra n g in g from 1 mg/ml t o 2 0 mg/ml were run s im u lta n e o u s ly .
I s o l a t i o n and p u r i f i c a t i o n o f th e p e p tid e m o ie ty o f t h e SA-
l i p o p r o t e i n
A lth o u g h S A - lip o p r o te in s were i s o l a t e d from t h r e e d i f
f e r e n t s p e c ie s o f mammals, th e p h y s i c a l and c h e m ic a l c h a r a c
t e r i z a t i o n of i t s p e p tid e m o ie ty was c a r r i e d o u t o n ly w ith
l i p o p r o t e i n s i s o l a t e d from cow lu n g s .
41
A. D e l i p i d a t i o n ■
S in c e t h e m o st i m p o r t a n t t o p i c t o he s t u d i e d i n t h e p r e
s e n t r e s e a r c h p ro b le m i s t h e i s o l a t i o n an d c h a r a c t e r i z a t i o n
o f t h e p e p t i d e m o ie ty o f t h e p u lm o n a ry s u r f a c e - a c t i v e l i p o
p r o t e i n co m p lex a m a jo r e m p h a s is h a s b een g iv e n t o t h e p ro b le m
o f s e p a r a t i n g l i p i d s fro m t h e l i p o p r o t e i n w i t h o u t d e n a t u r a t i n g
t h e p e p t i d e r e s i d u e . C o m p a ra tiv e d e l i p i d a t i o n s t u d i e s w ere
c a r r i e d o u t u t i l i z i n g t h e m eth o d s p r o p o s e d by F o l c h , e t a l .
( 1 9 5 7 ) , t h e m o d i f i c a t i o n o f S can u (1 9 6 6 ) t o t h e t e c h n i q u e '
p r e v i o u s l y p r o p o s e d by S c a n u , e t a l . ( 1 9 5 8 ) , and a new m eth o d
d e v e lo p e d by u s i n o u r l a b o r a t o r y .
I n a c c o r d w i t h F o l c h t s m e th o d , one volum e o f l i p o p r o t e i n
s u s p e n s io n was e x t r a c t e d w i t h tw e n ty volum es o f a m i x tu r e 2 : 1
o f c h lo r o f o r m - m e th a n o l ( v / v ) , f o r one h o u r a t room t e m p e r a t u r e
W ith S c a n u Ts t e c h n i q u e , o n e volum e o f t h e l i p o p r o t e i n s u s p e n
s io n was e x t r a c t e 'd w ith f i f t y v o lu m es o f a m i x t u r e 3 : 2 o f
e t h a n o l - e t h e r ( v /v ) a t - 1 0 ° f o r s i x h o u r s , f o llo w e d by a
se c o n d e x t r a c t i o n f o r e i g h t e e n h o u r s a t - 1 0 ° w ith t h e same
a l c o h o l - e t h e r m i x t u r e .
P r e l i m i n a r y s t u d i e s h a d shown t h a t i t was n e c e s s a r y t o
u t i l i z e some o t h e r d e l i p i d a t i o n p r o c e d u r e i n o r d e r t o rem ove
a l l t h e l i p i d s fro m t h e S A - l i p o p r o t e i n m a t e r i a l s . T h u s, t h e
f o llo w in g te c h n iq u e was d e v e lo p e d . One volume of t h e SA-
l i p o p r o t e i n s u s p e n s io n was e x t r a c t e d w ith t w e n t y - f i v e volum es
of a m ix tu re 3:2 o f e t h a n o l - d i e t h y l e t h e r ( v /v ) in a f l a t -
b o tto m , g l a s s - s t o p p e r e d f l a s k a t v ery low te m p e r a tu r e . The i
f l a s k c o n t a i n i n g t h e m ix tu re was c o o le d t o -7 0 ° by im m ersing
i t in a c o n t a i n e r w ith 2 - e t h o x y e th a n o l and d ry i c e , f o r f o u r j
h o u r s . The m ix tu re was shaken g e n tly by means of an a u to - j
m a tic s h a k e r (E b erb a c h C o r p o r a tio n , Ann A rb o r, M ichigan) o p er-i
a te d a t v e ry low s p e e d . The c o o le d l i p o p r o t e i n - o r g a n i c s o l
v e n t m ix tu re was th e n c e n t r i f u g e d a t 1 5 ,0 0 0 r .p .m . f o r t h i r t y |
m in u te s a t +4 in a S e r v a l l c e n t r i f u g e , u t i l i z i n g 50 ml capped}
!
p o ly e th y le n e t u b e s . The p r e c i p i t a t e was su sp en d ed in a f r e s h j
I
3:2 e t h a n o l - e t h e r s o l u t i o n ( f i f t y volumes o f o r g a n ic s o lv e n t I
j
f o r e a c h volume o f p r e c i p i t a t e ) and a llo w e d t o s ta n d f o r
tw e n t y - f o u r h o u rs a t - 2 0 ° in t h e f r e e z e r . The d e l i p i d i z e d
l i p o p r o t e i n was r e c o v e re d by c e n t r i f u g a t i o n a t 15,000 r .p .m . j
f o r t h i r t y m in u te s a t + 4 ° . The p r e c i p i t a t e d p e p tid e m a t e r i a l j
was th e n w ashed t h r e e tim e s w ith t h e e t h a n o l - e t h e r s o l u t i o n ,
and f i n a l l y t h r e e tim e s w ith d i e t h y l e t h e r in o r d e r t o remove I
a l l t r a c e s o f a l c o h o l from t h e d e l i p i d i z e d l i p o p r o t e i n , s in c e
i t h a s been o b s e rv e d t h a t r a p i d d e n a t u r a t i o n may r e s u l t from
i
I
i
t h e p r e s e n c e o f a l c o h o l in t h e p e p tid e r e s i d u e (S c a n u , e t a l
43
1 9 5 8 ). The p r e c i p i t a t e was a llo w e d t o d r y u nder n itr o g e n and
^ s t o r e d a t - 1 0 ° u n t i l u s e d .
D e l i p i d i z e d m a t e r i a l s o b ta in e d by t h e t h r e e d i f f e r e n t
te c h n iq u e s were a n a ly z e d f o r th e p r e s e n c e of p h o s p h o lip id s ;
and c h o le s te r o l...
B . S o l u b i l i t y of t h e d e l i p i d i z e d S A - lip o p r o te in :
S o l u b i l i t y s t u d i e s o f t h e d e l i p i d i z e d S A - lip o p r o te in w ere i
c o n d u c te d a t +4° and a t 24° (room te m p e r a tu r e ) in 0 .1 M sodium,
c h l o r i d e s o lu t i o n a t pH 5 .0 , 7 .0 , and 1 0 .0 ; in 0 .1 N sodium
h y d r o x id e ; and in d i s t i l l e d w a te r . S o l u t e and s o lv e n t s were
s t i r r e d f o r t w e n ty - f o u r h o u rs in an a u to m a tic m a g n e tic s t i r r e r
(P y ro -M a g n e tic , L a b -L in e I n s tr u m e n ts , I n c o r p o r a t e d , M elro se
P a rk , I l l i n o i s ) . Sam ples were c e n t r i f u g e d a t 1 5 ,0 0 0 r .p .m . I
f o r t h i r t y m in u te s a t +4° in a S e r v a l l r e f r i g e r a t e d - a u t o m a t i c
c e n t r i f u g e , and n i t r o g e n c o n te n ts o f t h e s u p e r n a ta n ts were '
d e t e r m in e d . ;
To d e te rm in e w h e th e r t h e s o l u b i l i z e d S A -p e p tid e was |
d i a l y z a b l e o r n o t , a 0 .5 p e r c e n t s o l u t i o n was p r e p a re d and I
d i a l y z e d a g a i n s t 0 .1 M sodium c h l o r i d e , pH 1 0 .0 , in a c o ld |
j
room a t + 4 ° . N itro g e n c o n te n ts o f th e d e l i p i d i z e d sam p le,
b e f o r e and a f t e r d i a l y s i s ., and o f th e d i a l y s a t e were d e t e r
m ined .
44
C. P u r i f i c a t i o n o f S A -p e p tid e
The cru d e p e p tid e s o lu t i o n s - were s u b je c te d t o a s e r i e s of
p u r i f i c a t i o n t e c h n iq u e s . The d r ie d d e l i p i d i z e d l i p o p r o t e i n
was re n d e re d s o lu b le by s t i r r i n g i t a t room te m p e r a tu re w ith
0 .1 N sodium h y d ro x id e f o r tw e n ty - f o u r h o u r s . T hese c ru d e
p e p tid e s o lu t i o n s w ere c o n c e n tr a te d and u l t r a f i l t e r e d u n d er
p r e s s u r e o v e r a D ia f lo Membrane, ty p e UM-1 ( D ia f lo U l t r a f i l
t r a t i o n , Amicon C o r p o r a tio n , C am bridge, M a s s a c h u s e tts ) . T h is
membrane r e t a i n s s o l u t e s l a r g e r th a n 1 0 , 0 0 0 m o le c u la r w e ig h t.
The u l t r a f i l t e r e d , c o n c e n tr a te d S A -p ep tid e s o lu t i o n s were
th e n s u c c e s s i v e l y g e l f i l t e r e d th ro u g h columns o f Sephadex
i
j
G -20, G -50, and G-100 (P h arm acia F in e C h em ic a ls, I n c o r p o r a te d ,I
U p p sa la , Sweden) o f medium p a r t i c l e s i z e . G el f i l t r a t i o n s j
were c a r r i e d o u t w ith columns of s i z e 2 .5 x 4 .5 cm, and t h e i
Sephadex g e l s were e q u i l i b r a t e d w ith 0 .1 M sodium c h l o r i d e ,
pH 1 0 .0 . The S A -p e p tid e s o lu t i o n s were p la c e d on th e colum ns j
I
I
and e l u t i o n s were p e rfo rm ed w ith th e same s a l i n e s o l u t i o n s . ; ■
i
i
R ecovery o f t h e p e p tid e was d e te rm in e d by m e a su rin g t h e l i g h t r
I
a b s o r p tio n a t 280mu o f th e c o l l e c t e d f r a c t i o n s a n d /o r by d e - j
te r m in in g t h e p re s e n c e of p r o te in by a m ic r o b iu r e t m ethod
i
( A l l e r t o n , 1 9 6 7 ). A l l t h e e l u t e d f r a c t i o n s c o n ta in in g p r o t e i n |
were p o o led t o g e t h e r , l y o p h i l i z e d , and r e d i s s o lv e d w ith |
45
d i s t i l l e d w a te r . The f i n a l p e p t i d e s o l u t i o n c o l l e c t e d a f t e r
g e l f i l t r a t i o n th r o u g h S ephadex G-100 was e x t e n s i v e l y d ia ly z e d
a g a i n s t 0 .1 M sodium c h l o r i d e , pH 1 0 .0 , f o r f o u r days in t h e ;
c o ld room, w ith ch an g es of d i a l y s a t e e v e ry tw e n ty - f o u r h o u r s . I
The d i a l y z e d S A -p e p tid e s o l u t i o n s w ere exam ined spec--
i t r o p h o t o m e t r i c a l l y o v e r t h e ra n g e o f v i s i b l e and u l t r a v i o l e t ’
■ i
l i g h t (fro m 800 t o 200mjj.) in a Beckman DB S p e c tr o p h o to m e te r , i
in o r d e r t o d e t e c t a b s o r p tio n p e a k s o t h e r th a n t h a t of p u re
p r o t e i n s a t 280mp.. :
D. S A -p e p tid e c o n te n t o f t h e s a l i n e s o l u t i o n s
The d ry w e ig h t o f t h e m a t e r i a l t o be used in t h e p h y s i c a l ' ■
! I
! and c h e m ic a l e x p e rim e n ts was d e te rm in e d in t h e same way as
i • i
! j
d e s c r ib e d in s e c t i o n I , B. A liq u o ts o f 5 0 ,0 ml e a c h o f t h e j
• d i a l y z e d S A -p e p tid e s o l u t i o n s w ere u sed i n t h e s e e x p e r i m e n t s .
I I I . C hem ical c h a r a c t e r i z a t i o n o f t h e S A -p e p tid e m o ie ty
In o r d e r t o s tu d y t h e c h e m ic a l c o m p o sitio n o f t h e SA- j
I I
| p e p t i d e , a s e r i e s o f c h e m ic a l t e s t s w ere p e rfo rm e d w ith i t . j
A. D e te rm in a tio n o f p r o t e i n c o n te n t |
; 1
i • ;
S The p r o t e i n c o n te n t o f t h e S A -p e p tid e m o ie ty was e s t a b - |
3 ' I
] ' j
l i s h e d by e i t h e r m e a s u rin g i t s t o t a l n i t r o g e n c o n t e n t , a n d / o r I
j
by u se o f t h e m ic r o b i u r e t m eth o d . !
46 ;
1 . T o t a l n i t r o g e n . T echnicon M ic r o - K je ld a h l (1965)
T h is m ethod i s t h e same a s d e s c r ib e d in s e c t i o n I ,
c . i . ;
2* P r o t e i n c o n t e n t . M ic r o b iu r e t ( A l l e r t o n , 1967)
T h is m ethod was u se d b e c a u se of i t s h ig h s e n s i - j
i
t i v i t y . A b i u r e t r e a g e n t was p r e p a re d f r e s h by a d d in g d r o p - |
^ i
w is e , w ith s t i r r i n g , 200 ml o f 1 p e r c e n t CuSO^ • 5H20 (M ai- j
l i n c k r o d t ) t o one l i t e r o f 40 p e r c e n t NaOH. One ml o f t h i s ;
b i u r e t r e a g e n t was added t o 1 .0 ml o f d i s t i l l e d w a te r , and i
1 .0 ml o f p e p t i d e s o l u t i o n . Tubes w ere s t i r r e d and a llo w e d I
t o s ta n d f o r te n m in u te s ( S o lu tio n A ) . The o p t i c a l d e n s i t y i
i
I
was re a d a t 290nu and a t 390rri_t.. A s o l u t i o n c o n t a i n i n g 1 .0 ml ]
I
o f w a te r , 1 .0 ml o f 40 p e r c e n t NaOH, and 1 .0 m l of p e p t i d e j
|
s o l u t i o n was a l s o r e a d a t t h e same w a v e le n g th s ( S o lu tio n B ) . i
The r e a d in g s were s u b t r a c t e d from t h o s e g iv e n by sam ple co n
t a i n i n g b i u r e t re a g e n t," i . e . , A-B, a t b o th 290nu and a t 390mp,.
The d i f f e r e n c e o b ta in e d when t h e f i n a l r e a d in g a t 390mu was
s u b s t r a c t e d from t h e f i n a l r e a d in g a t 290ny,, i s p r o p o r t i o n a l |
f
1
)
t o t h e num ber o f p e p t i d e bonds p r e s e n t . . A s e r i e s o f d i l u t i o n j
s ta n d a r d s o f album in (B o v in e P lasm a M e rc a p ta lb u m in , p ro v id e d
by D r. Sam uel E. A l l e r t o n , B io p h y s ic s L a b o ra to r y ) r a n g in g fromj
|
50ug/m l t o 500(j,g/ml w ere run s im u lta n e o u s ly f o r co m p ariso n . j
47
A b la n k c o n ta in in g d i s t i l l e d w a te r and 40 p e r c e n t NaOH was
in c lu d e d in each ru n . N ote: The t o t a l n itro g e n c o n te n t of
th e album in s ta n d a rd was a l s o d e te rm in e d by th e m ic ro -K je ld a h l
m ethod, in o rd e r t o e s t a b l i s h c o r r e c t l y i t s p r o te in c o n te n t. I
i
B . L ip id a n a l y s is :
I n v e s t i g a t i o n s f o r 't h e p re se n c e of l i p i d s in t h e SA- j
}
p e p tid e m o iety were done by th e m ethods d e s c rib e d under s e c
t i o n I , C .2 .
C . C a rb o h y d ra te a n a l y s is
The f o llo w in g c o l o r im e t r i c t e s t s were u t i l i z e d t o d e t e c t j
t h e p re se n c e of c a rb o h y d ra te s in th e S A -p e p tid e . ;
1, S i a l i c a c id (N -a c e th y l-N e u ra m in ic A c i d ) . Warren
|
(1959) |
I
T h is method r e q u i r e s m ild h y d r o ly s is p r i o r t o th e j
c o l o r i m e t r i c a s s a y . One ml of th e sam ple was d i l u t e d in 1 ml ’
o f 0 .2 N HgSO^ and in c u b a te d in an 80° w a te r b a th f o r one j
i
h o u r .. To d e te rm in e th e c o n c e n tr a tio n of f r e e s i a l i c r a c id ,. j
Q.20 ml o f th e h y d r o ly s a te was in c u b a te d w ith 0.10 ml of 0 .2 M i
i
sodium p e r i o d a t e (M a th e so n ). T his r e a c tio n was sto p p e d a f t e r j
e x a c t l y 1,200 seconds by th e a d d itio n of 1 .0 ml of 10 p e r
c e n t sodium a r s e n i t e (M ath eso n ), th e n 3.0 ml of 0 .6 p e r c e n t j
i
t h i o b a r b i t u r i c a c id (M atheson) was added. The s o lu tio n was j
48
in c u b a te d in a b o i l i n g w a te r b a t h f o r e x a c t l y 1 ,2 0 0 s e c o n d s ,
c o o le d and t h e c o l o r d e v e lo p e d was e x t r a c t e d w ith 4 .3 ml
c y c lo h e x a n o n e ( M a l l i n c k r o d t ) . The o p t i c a l d e n s i t y was re a d
a t 549 mu,, in a Beckman DB S p e c tr o p h o to m e te r , and com pared
w ith v a lu e s o b ta in e d f o r a d i l u t i o n s e r i e s o f s ta n d a r d s i a l i c
a c i d ( N -a c e th y l- N e u r a m in ic A c id , C a lb io c h e m , A g r a d e ) , from
2 5 u g /m l t o 2 0 0 u g /m l. A r e a g e n t b la n k was in c lu d e d in e a c h
e x p e r i m e n t .
2 . F u c o se (M e th y l p e n t o s e ) . D isc h e and S h e t t l e s
(1 9 4 8 ) as p ro p o s e d by D is c h e (1962)
T h is m ethod was em ployed w ith o u t p r i o r h y d r o l y s i s
To 4 .5 m l c o ld HgSO^ r e a g e n t ( p r e p a r e d by a d d in g s i x p a r t s o f
c o n c e n t r a t e d s u l f u r i c a c i d t o one p a r t o f w a te r) was added
1 .0 m l o f th e sa m p le . The s o l u t i o n was b ro u g h t s lo w ly t o
room t e m p e r a t u r e , in c u b a te d i n a b o i l i n g w a te r b a th f o r te n
m in u te s and r e t u r n e d q u ic k ly t o room t e m p e r a tu r e in a 20°
w a te r b a t h . E x a c t l y 0 .1 0 m l o f 3 p e r c e n t aq u eo u s s o l u t i o n
o f c y s t e i n e (C a lb io c h e m , A g ra d e ) h y d r o c h lo r id e was a d d e d .
A f t e r a tw o -h o u r w a it f o r c o l o r d e v e lo p m e n t, t h e o p t i c a l d e n
s i t y a t 427mu was s u b t r a c t e d from t h a t a t 396m|j,. S ta n d a r d s
o f L - f u c o s e (C a lb io c h e m , A g r a d e ) . r a n g in g from 2 5 u g /m l t o
100|_ig/ml w ere run s im u lta n e o u s ly and u sed f o r c o m p a riso n . A
49
r e a g e n t b la n k was i n c l u d e d in e a c h e x p e r i m e n t .
3 . N e u t r a l H e x o s e s , O r c i n o l T e s t . ' L u s t i g and L a n g e r
(1 9 3 1 ) a s m o d ifie d by F r a n c i s , e t- a l . (1 9 6 2 )
T h is m ethod was u sed w ith o u t p r i o r h y d r o l y s i s o f
t h e s a m p l e s . An O r c i n o l - s u l f u r i c a c i d r e a g e n t was p r e p a r e d
f r e s h fro m tw o p a r t s o f 1 .6 p e r c e n t O r c i n o l (C a lb io c h e m , C
g r a d e ) and f i f t e e n p a r t s o f 60 p e r c e n t s u l f u r i c a c i d . Ex
a c t l y 5 .2 5 m l o f t h i s c h i l l e d r e a g e n t was ad d ed t o 0 .5 0 ml o f
t h e s a m p le . The t u b e s w ere b r o u g h t t o room t e m p e r a t u r e , i n
c u b a te d i n an 80° w a te r b a t h f o r f i f t e e n m in u te s , t h e n c o o le d
q u i c k l y t o room t e m p e r a t u r e . The o p t i c a l d e n s i t y was r e a d a t
505ny,. A d i l u t i o n s e r i e s o f m annose (C a lb io c h e m , A g r a d e )
o r g a l a c t o s e (C a lb io c h e m , A g ra d e ) from 25|ug/m l t o 200(ig/m l,
was u se d f o r c o m p a r is o n . A r e a g e n t b la n k was i n c l u d e d in
e a c h e x p e r i m e n t .
4 . H e x o s a m in e s . M o d if i c a t i o n o f t h e E lso n -M o rg an
T e s t by R o n d le an d Morgan (1 9 5 5 )
T h is m ethod was u sed f o r t h e d e t e r m in a ti o n o f t o
t a l h e x o s a m in e . The n e c e s s a r y p r i o r h y d r o l y s i s was accom p
l i s h e d by a t h r e e - h o u r i n c u b a t i o n a t 100° in 3 .6 0 N HC1 (tw o
p a r t s o f sam p le in t h r e e p a r t s o f c o n s t a n t b o i l i n g HC1) . The
h y d r o l y s a t e was d r i e d o v e r NaOH p e l l e t s in v a c u o , and
50 |
r e d is s o lv e d in 2 .0 ml o f d i s t i l l e d w a te r. To 1 .0 ml o f t h i s
sam ple was added 2 .0 ml of a c e ty la c e to n e re a g e n t (p re p a re d
f r e s h from one p a r t of a c e ty la c e to n e (M atheson) and tw e n ty - ;
f i v e p a r t s of 1 N sodium c a rb o n a te ( M a llin c k r o d t) , fo llo w e d j
i
i
by in c u b a tio n f o r - tw e n ty m in u tes in a b o i l i n g w a te r b a th , and ;
c o o lin g t o room te m p e r a tu re . E x a c tly 6 .0 ml o f e th a n o l and j
|
1 .0 ml of E h r l i c h ’s re a g e n t (p re p a re d f r e s h from 0 .8 gm of
p -d im eth y lam in o b en zald eh y d e (M atheson), 15 ml o f e t h a n o l, and 1
15 ml of c o n c e n tr a te d HC1) were added. The tu b e s were in c u - j
b a te d f o r te n m in u tes a t 68° ( tim in g was s t a r t e d when t h e
te m p e ra tu re i n s i d e t h e tu b e s reach ed 6 8 ° ), and th e n b ro u g h t !
(
t o room te m p e ra tu re a g a in . The o p t i c a l d e n s it y was re a d a t j
f
530mp. and compared t o a d i l u t i o n s e r i e s o f s ta n d a rd s o f g lu c o
sam ine (C albiochem , A grad e) ra n g in g from 25(_ig/ml t o 200u,g/ml.:
A re a g e n t b la n k was in c lu d e d in each e x p e rim e n t.
D. E s ta b lis h m e n t of t h e p e p tid e - c a r b o h y d r a te m oiety e x is te n c e
In o r d e r t o e s t a b l i s h t h a t th e p re se n c e o f c a rb o h y d ra te s j
s
in t h e p e p tid e s o lu t i o n s co u ld n o t a r i s e from a p r o te in im- j
!
p u r i t y o f h ig h c a rb o h y d ra te c o n te n t, th e p u r i f i e d S A -p ep tid e
I
I
was s u b je c t t o p ro c e d u re s d e sig n e d t o s e p a r a te t h e p e p tid e j
from known g l y c o p r o te in s . The c a rb o h y d ra te c o n te n t ( s i a l i c j
i
i
a c id ) of t h e r e i s o l a t e d S A -p ep tid e was th e n compared w ith t h a t j
51
of t h e s t a r t i n g m a t e r i a l .
T re a tm e n t o f t h e S A -p e p tid e w ith 5 p e r c e n t t r i c h l o r o
a c e t i c a c id o r 0 .6 M p e r c h l o r i c a c id was em ployed. The p ep
t i d e was p r e c i p i t a t e d by a d d in g 1 .0 ml o f 10 p e r c e n t t r i - !
j
c h l o r o a c e t i c a c id ( M a l l i n c k r o d t) , o r 1 .2 M p e r c h l o r i c a c id
( p re p a r e d by d i s s o l v i n g 172.22 ml of 70 p e r c e n t p e r c h l o r i c j
a c id ( M a llin c k r o d t) in d i s t i l l e d w a te r in an 1 l i t e r v o lu m et- ;
r i c f l a s k ) t o 1 .0 ml o f S A -p e p tid e s o l u t i o n . P r e c i p i t a t e ;
was c o l l e c t e d by c e n t r i f u g a t i o n , washed and re su sp e n d e d in j
1 .0 ml of d i s t i l l e d w a te r . M easurem ent of t h e s i a l i c a c id j
c o n te n t was th e n p erfo rm ed a c c o r d in g t o Warren (1 9 5 9 ), T hese j
m ethods were chosen b e c a u se of t h e re c o g n iz e d s o l u b i l i t y of j
many known g ly c o p r o te in s un d er t h e s e c o n d i t i o n s , w hich p r o - I
i
d u ces p r e c i p i t a t i o n of o t h e r p r o t e i n s p e c ie s ( S c h u ltz e , 1962).:
E . Amino a c id a n a l y s i s 2
D e te rm in a tio n o f t h e amino a c i d c o m p o sitio n o f t h e SA- j
t
}
p e p tid e i s o f param ount im p o rta n c e in t h e p r e s e n t r e s e a r c h . i
One m l of c o n c e n tr a te d h y d r o c h lo r ic a c id (12 N) was added t o j.
I
1 .0 t o 4 .5 mg o f d e l i p i d a t e d S A - lip o p r o te in in 1 ml w a te r j
s o l u t i o n ; t h e g l a s s tu b e s were e v a c u a te d , s e a le d under
2
We a r e in d e b te d t o Mr. Kim S. Wise (L a b o ra to ry T e c h n i
c ia n , D epartm ent o f B io c h e m is try , U n iv e r s ity o f S o u th ern C a l i f
o r n ia D e n ta l S ch o o l) f o r h i s h e lp in p e rfo rm in g th e ru n s w ith
t h e amino a c i d 'a n a l y z e r . _________ _______________________
52
n i t r o g e n , and h e a te d a t 110 ± 1 ° f o r 22, 4 6 , o r 72 h o u r s .
H y d r o ly s a te d w ere d r i e d o v e r NaOH p e l l e t s in v a c u o . The
d ry sam p les w ere d i s s o l v e d in 2 .0 m l 0 .2 0 N sodium c i t r a t e -
HC1 b u f f e r , pH 2 . 2 , A l iq u o ts w ere a n a ly z e d by io n ex ch an g e
c h ro m a to g ra p h y w ith t h e te c h n iq u e o f M oore, Spackm an, and
S t e i n (1 9 5 8 ), in a B eckm an/S pinco m odel 120B amino a c i d a n a l y
z e r (Spackm an, e t _ a l., 1 9 5 8 ). The a n a l y z e r was c a l i b r a t e d
w ith t h e t y p e 1 am ino a c i d c a l i b r a t i o n m ix tu r e ( p ro v id e d by
Beckman I n s t r u m e n t s , I n c o r p o r a t e d , P a lo A l t o , C a l i f o r n i a ) a n d ,
in s t u d i e s o f p e r f o r m a t e - o x i d i z e d p e p t i d e , w ith c y s t e i c a c i d .
C h ro m ato g rap h y was done a c c o r d in g t o t h e p r o c e d u re f o r . a
" S ta n d a rd 50° ru n " (Spackm an, 1 9 6 2 ).
H a l f - c y s t i n e was d e te r m in e d a s c y s t e i c a c i d a f t e r o x i
d i z i n g t h e S A -p e p tid e w ith p e r f o r m ic a c i d a c c o r d in g t o th e
m ethod o f Schram , e t _al. (1 9 5 4 ), and Moore (1 9 6 3 ), a s recom
mended by B a i l e y (1 9 6 7 ). A p e r f o r m ic a c i d r e a g e n t was p r e
p a r e d f r e s h from n in e p a r t s o f 88 p e r c e n t f o rm ic a c i d (M al
l i n c k r o d t ) and one p a r t o f 30 p e r c e n t h y d ro g en p e r o x i d e .
E x a c tly 2 .0 m l o f t h i s c h i l l e d r e a g e n t was ad d ed t o t h e p e p
t i d e sam ple c o n t a i n i n g 2 t o ' 4 mg o f p r o t e i n . The m ix tu r e was
a llo w e d t o s ta n d f o u r h o u r s a t 0 ° . Then 0 .3 0 ml o f 48 p e r
c e n t h y d ro b ro m ic a c i d was added and t h e s o l u t i o n was m ixed
and d r ie d a t 40° jLn v acu o . R esid u e was th e n h y d ro ly z e d in the;
i
|
u s u a l way w ith 6 N HC1 a t 110 + 1° f o r tw en ty -tw o h o u rs, and j
j
a l i q u o t s were a n aly zed by ion exchange chrom atography w ith j
5
th e Beckman/Spinco model 120B amino a c id a n a ly z e r .
j
F. C o lo r im e tric d e te r m in a tio n o f t r y p to p h a n . S p ie s and j
Chambers (1949) j
i
The try p to p h a n c o n te n t o f th e i n t a c t S A -p ep tid e was d e te r-;
mined a c c o rd in g t o p ro c e d u re L of t h e method pro p o sed by Spies!
and Chambers (1 9 4 9 ). R e a c tio n s, were c a r r i e d out a t room tern- j
p e r a t u r e and p r o te c te d from l i g h t . To 30 mg of s o l i d p - d i - ■ j
m ethylam inobenzaldehyde (M atheson) was added 9 .0 ml of 2 1 .4 N ;
s u l f u r i c a c id , d is s o lv e d and co o le d t o room te m p e r a tu re . To !
i
I
t h i s s o lu tio n 1 .0 ml o f t h e p e p tid e sam ple was added, m ixed, j
i
j
c o o le d t o room te m p e ra tu re and allo w ed t o s ta n d f o r tw e n ty - j
f o u r h ours in t h e d a rk . To t h i s m ix tu re th e n was added 0.10
ml of an 0.045 p e r c e n t s o lu t i o n o f sodium n i t r i t e . The s o l
u tio n was mixed and allo w ed t o s ta n d f o r t h i r t y m in u tes in
t h e d a rk . The o p t i c a l d e n s it y was re a d a t 590jh |_ l and t r a n s
m it an cy v a lu e s were c o n v e rte d t o w eig h t o f try p to p h a n from
s ta n d a rd c u rv e C of S p ie s and Chambers (1 9 4 8 ). A b la n k
s o lu t i o n was run e ach tim e w ith t h e p e p tid e s o lu tio n b u t
p -d im eth y lam in o b en zald eh y d e was n o t u se d .
54 j
|
G . C a l c u l a t i o n o f amino a c id c o m p o sitio n i
i
D a ta from t h e A n a ly z e r w ere c o r r e c t e d f o r l o s s e s of u n - j
!
s t a b l e amino a c id s d u r in g a c id h y d r o l y s i s . V alues o f g l u t a - j
i
m ic a c i d , a s p a r t i c a c i d , l y s i n e , h y d r o x y ly s in e , t h r e o n in e and j
i
i
s e r i n e w ere o b ta in e d by e x t r a p o l a t i o n t o z e r t o tim e of h y d ro - :
l y s i s t o a c c o u n t f o r a p p a r e n t l o s s o f t h e s e com pounds. The j
am ide ammonia c o n te n t was a l s o e s tim a te d by e x t r a p o l a t i o n t o j
j
z e r o tim e o f h y d r o l y s i s . V alu es f o r t h e c o n te n t o f h a l f - ;
c y s t i n e and tr y p to p h a n w ere o b ta in e d by m ethods a lr e a d y d e s - j
t
I
c r i b e d . The m oles o f e a c h amino a c id r e s i d u e w ere c o n v e rte d i
i
i n t o grams of am ino a c id r e s i d u e p e r 100 grams o f p e p t i d e . |
j
The number o f m oles o f e a c h am ino a c i d p r e s e n t in 100,000 j
grams of p e p tid e was a l s o c a l c u l a t e d , and from t h e s e v a lu e s j
m in im al m o le c u la r w e ig h ts o f t h e p e p tid e m o ie ty w ere computed.!
I
The n i t r o g e n v a lu e f o r e ach amino a c id r e s i d u e s was a l s o c a l - j
c u l a t e d a s p e r c e n t of t o t a l n i t r o g e n . :
P h y s i c a l c h a r a c t e r i z a t i o n of t h e S A -p e p tid e m o ie ty i
E x p e rim e n ts w ere p e rfo rm ed in t h e B io p h y s ic s L a b o ra to r y :
t o e s t a b l i s h t h e p h y s i c a l c h a r a c t e r i s t i c s o f t h e p e p t i d e j
j
m o ie ty o f t h e S A - lip o p r o te in com plex. j
A . C a l c u l a t i o n o f t h e p a r t i a l s p e c i f i c volume
The p a r t i a l s p e c i f i c volume o f' a p r o t e i n can be e s tim a te d |
I
q u i t e r e l i a b l y from i t s c h e m ic a l c o m p o s itio n , s in c e i t h a s j
55 I
been found t h a t th e volum es of m acro m o lecu les a r e c l o s e l y
e q u a l t o th e sum o f t h e i n d i v i d u a l r e s id u e c o n t r ib u t i o n s
I
(McMeekin, e t a l . , 1949; McMeekin and M a rs h a ll, 1952; ;
i
j
Schachman, 1 9 6 5 ). In th e c a l c u l a t i o n of th e p a r t i a l s p e c i f i c I
volume o f th e S A -p e p tid e t h i s method o f a d d in g t h e i n d i v i d u a l j
I
s p e c i f i c volumes of th e r e s id u e s t h a t c o n s t i t u t e i t was u se d , j
j
The v a lu e s f o r t h e s p e c i f i c volumes o f t h e amino a c id r e s i - i
I
dues a r e th o s e g iv en by Cohn and E d s a l l (1 9 4 3 ), and t h e values!
j
f o r t h e s p e c i f i c volumes o f th e c a rb o h y d ra te r e s id u e s a r e i
I
i
th o s e g iv en by G ibbons (1 9 6 6 ). The s p e c i f i c volume o f hydrox>4
■ |
I
l y s i n e was c a l c u l a t e d from l y s i n e Vi v a lu e a c c o r d in g t o Cohn |
i
and E d s a l l (1 9 4 3 ), assum ing t h e v a lu e o f 2 .3 c c/m o le f o r t h e |
e x t r a atom o f oxygen p r e s e n t in h y d r o x y ly s in e . In t h e c a lc u - j
l a t i o n of t h e p a r t i a l s p e c i f i c volume o f th e S A -p e p tid e , t h e j
i
fo llo w in g fo rm u la was u sed :
V = £W i V i/ £ Wi
where " V i s t h e p a r t i a l s p e c i f i c volume o f t h e p r o t e i n , and j
i
Wi and Vi a re t h e v a lu e s in grams p e r c e n t and th e s p e c i f i c j
I
volumes f o r each i n d i v i d u a l r e s id u e , r e s p e c t i v e l y .
B • E l e c tr o p h o r e s i s i
I
j
The c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s te c h n iq u e of B r i e r e j
i
and M u ll (1 9 6 6 ), w ith a co m m ercial a p p a ra tu s . (Gelman I n s t r u -
t
ment Company, Wagner Road, Ann A rb o r, M ichigan) was u se d . A j
56
Beckman m odel RB A n a l y t r o l (Beckman I n s tr u m e n ts I n c o r p o r a te d ,
i
I
P a lo A lto , C a l i f o r n i a ) was a d a p te d t o scan t h e c e l l u l o s e a c e - ;
t a t e s t r i p s (Gelman) by means o f th e a d d i t i o n of an e x t e r n a l j
i
sy n chronous m otor ( a S c a n -A -tro n , Gelman) w hich drew t h e I
\
s t r i p s th ro u g h a t t h e r a t e o f tw o in c h e s p e r m in u te . The j
n a rro w e s t s l i t in t h e A n a ly tr o l was s h o rte n e d t o 1 cm in ■ j
l e n g t h w ith a b la c k m asking t a p e . The B-2 o p t i c a l d e n s i t y j
!
cam was u sed w ith t h e green 500m)j, f i l t e r . I
I
I
A 1 p e r c e n t s o lu t i o n o f t h e S A -p e p tid e was p r e p a re d j
1
and d ia ly z e d f o r t h r e e days a g a i n s t B a r b i t a l - b a r b i t u r i c a c id j
b u f f e r , pH 8 .6 , i o n i c s t r e n g t h 0 .0 5 ( p re p a r e d by d i s s o l v i n g I
I
I
1 0 .3 0 gm o f sodium d i e t h y l b a r b i t u r i c a c id (Merck) and 1 .8 4 gm|
I
j
o f d i e t h y l b a r b i t u r i c a c id (Merck) in 1 l i t e r of d i s t i l l e d j
w a te r) w ith d a i l y changes o f d i a l y s a t e . E l e c tr o p h o r e s i s was |
p e rfo rm ed on t h e s e d ia ly z e d m a t e r i a l s . A s m a ll sam ple (2 t o i
3 lam bdas) was a p p lie d t o th e c e l l u l o s e a c e t a t e s t r i p s ( p r e - |
soaked in t h e same b a r b i t a l b u f f e r ) a t a b o u t two in c h e s from j
one end o f t h e s t r i p s (on t h e c a th o d e s id e o f th e m i d l i n e ) . ;
i
The s t r i p s were im m e d ia te ly t r a n s f e r r e d t o t h e e le c tr o p h o r e s is !
chamber c o n ta in in g b a r b i t a l b u f f e r , w ith o n ly t h e i r ends d i p - |
. ' i
p in g i n t o t h e b u f f e r . E l e c tr o p h o r e s i s was c a r r i e d o u t a t !
i
i
1 .5 ma p e r s t r i p . f o r t h i r t y m in u te s . When t h e run was o v e r I
i
I
I
I
I
t h e s t r i p s w ere rem oved and s t a i n e d f o r f i v e m in u te s w ith
P o n ceau S ( p r e p a r e d by d i s s o l v i n g 0 .0 2 gm o f P o n c e a u S j
i
( A l l i e d C h e m ic a ls ) i n 100 m l o f 15 p e r c e n t t r i c h l o r o a c e t i c 1
a c i d ) , and t r e a t e d w ith r i n s e s o f 5 p e r c e n t a c e t i c a c i d in
w a te r u n t i l t h e b a c k g ro u n d s t a i n was rem oved. The s t r i p s
w ere th e n r e s t a i n e d f o r f i v e m in u te s w ith N ig r o s i n ( p r e p a r e d j
I
by d i s s o l v i n g 0 .0 2 gm o f N ig r o s i n ( A l l i e d C h e m ic a ls ) in 100 ml!
I
o f d i s t i l l e d w a t e r ) . E x c e ss o f b a c k g ro u n d was rem oved by j
l
t
m eans o f r i n s e s w ith d i s t i l l e d w a t e r . S t r i p s w ere d r i e d by j
1
p l a c i n g them b etw een s e v e r a l l a y e r s o f b l o t t i n g p a p e r . The j
d r i e d s t r i p s w ere c l a r i f i e d by im m e rsin g them i n t o a s o l u t i o n I
o f 10 p e r c e n t a c e t i c a c i d in m e th a n o l f o r t h i r t y t o s i x t y
s e c o n d s , u n t i l c o m p le te ly w e t. S t r i p s becam e t r a n s p a r e n t as
t h e y d r i e d . The t r a n s p a r e n t s t r i p s w ere sc a n n e d by u s i n g t h e j
|
i
A n a l y t r o l R e c o rd in g D e n s ito m e te r I n t e g r a t o r (B eck m an ). j
C . A n a l y t i c a l U l t r a c e n t r i f u g a t i o n
i
S t u d i e s on t h e d e t e r m i n a t i o n ■ o f t h e m o le c u la r w e ig h t and j
s e d im e n t a t i o n c o e f f f i c i e n t s o f t h e S A -p e p tid e m o ie ty w ere p e r-I
i
i
fo rm ed by u t i l i z i n g u l t r a c e n t r i f u g a t i o n t e c h n i q u e s . U l t r a
c e n t r i f u g a t i o n ru n s w ere c o n d u c te d i n a M odel E . S p in c o A n a l y t
i c a l U l t r a c e n t r i f u g e (Beckman I n s t r u m e n ts I n c o r p o r a t e d , P a lo
A l t o , C a l i f o r n i a ) , o p e r a t e d a t room t e m p e r a t u r e i n t h e
58
B io p h y sic s L a b o ra to ry . The two h o le AN-D r o t o r was used and
i t s te m p e ra tu re was r e g u la te d t o w ith in +0. 1° d u rin g th e
e x p e rim en ts by th e te m p e ra tu re c o n t r o l u n i t . C e lls were
tig h t e n e d 2 -1 /2 d i v i s i o n s on a to rq u e w rench. The s c h lie r e n
o p t i c a l system was u sed th ro u g h o u t t h e s e e x p e rim e n ts. The
s c h li e r e n p a t t e r n s were p h o to g ra p h ed on Kodak m e ta llo g ra p h ic
p l a t e s which were d ev elo p ed w ith Kodak D-19, and were o t h e r
w ise p ro c e s s e d a c c o rd in g t o th e m a n u f a c tu re r ’s d i r e c t i o n s .
A Nikon Shadowgraph Model 6 C o p t i c a l co m p arato r (Nikon Com- j
pany, J a p a n ) , was u sed t o re a d t h e p h o to g ra p h ic p l a t e s . j
O p e ra tio n and c a re o f th e u l t r a c e n t r i f u g e was done a c c o rd in g ;
t o th e S p in co I n s t r u c t i o n M anual (1 9 6 1 ). j
1 . D e te rm in a tio n of se d im e n ta tio n c o e f f i c i e n t s I
S e d im e n ta tio n c o e f f i c i e n t s of t h e S A -p ep tid e com- j
p o n e n ts were d e te rm in e d by means of s e d im e n ta tio n v e l o c i t y j
|
e x p e rim e n ts. An a p p ro x im a te ly lg m /d l s o lu tio n of S A -p ep tid e j
i
/v £
was d ia ly z e d f o r f o u r h o u rs a t +4 a g a in s t 0 .1 i o n i c s tr e n g th ,!
i
i
pH 8 .0 , 0 .1 M sodium c h lo r id e s o l u t i o n , changed s e v e r a l tim e s |
d u rin g t h e d i a l y s i s . The p r o te in s o lu tio n was d i l u t e d w ith
f i n a l d i a l y s a t e t o g iv e a s o lu tio n o f c o n c e n tr a tio n 0 .7 6 g m /d l,
d e te rm in e d by t h e m ic ro b iu r e t a n d /o r th e m ic ro -K je ld a h l me
th o d s . A s e d im e n ta tio n v e l o c i t y run was p erfo rm ed w ith t h i s
59 !
I
s o l u t i o n a t 2 0 .8 5 ° , a c c o r d in g t o Schachman (1 9 6 5 ). A 4° s e c
t o r s ta n d a r d 12 mm alum inium c e n t e r p i e c e c e l l was u s e d w ith
a dummy c o u n te r b a la n c e in t h e tw o h o ld AN-D r o t o r . S even-
t e n t h s m l o f s o l u t i o n was p la c e d in th e c e l l c o m p a rtm e n t.
A f t e r t h e r o t o r had been c o u p le d t o t h e d r i v e and t h e cham ber
e v a c u a te d , t h e ru n was s t a r t e d . The s c h l i e r e n o p t i c a l sy stem
was u se d w ith t h e p h a s e p la te a t an a n g le o f 70° t o t h e v e r t i -
t
i
c a l . P h o to g ra p h s w ere ta k e n a u t o m a t i c a l l y 4 T, 2 0 T, 36*, 5 2 1, j
j
and 68* a f t e r t h e r o t o r had a t t a i n e d a f i n a l sp e e d o f 56,100 |
»
r .p . m . A g ree n l i g h t f i l t e r was em ployed w ith t h e m ercu ry
lamp s o u r c e . The p h o to g ra p h ic p l a t e s w ere p la c e d on t h e s ta g e
o f t h e c o m p a ra to r and r e a d . By m e a su rin g t h e d i s t a n c e he-,
tw een t h e i n s i d e ( r i g h t ) edge o f t h e l e f t b a r , and t h e peak s
o f t h e b o u n d ary (X o r d i n a t e ) , and d i v i d i n g by t h e h o r i z o n t a l j
m a g n i f i c a ti o n f a c t o r ( F ) , t h e r a d i a l p o s i t i o n o f e a c h peak was
c a l c u l a t e d . The i n s i d e ( r i g h t ) edge o f t h e l e f t r e f e r e n c e
b a r i s 5.7 0 0 0 cm from t h e c e n t e r o f r o t a t i o n .
S e d im e n ta tio n c o e f f i c i e n t s w ere c a l c u l a t e d by u s in g t h e j
e q u a tio n :
1 dx
3 = - n - — - (2 )
w^x d t
w here x i s t h e d i s t a n c e o f t h e boundary in c e n t i m e t e r s from j
t h e a x i s o f r o t a t i o n , t i s t h e tim e in s e c o n d s , an d w i s t h e
60
a n g u la r v e l o c i t y in r a d ia n s p e r seco n d .
C o rre c tio n s of th e o b serv ed s e d im e n ta tio n c o e f f i c i e n t s ,
Sobs ■ 5 t o th e s ta n d a rd s t a t e ( s - ^ « ) , were made a c c o rd in g t o
t h e e q u a tio n :
where C ^ t / j Q 20) i s th e v i s c o s i t y o f w a te r a t t ° r e l a t i v e to
t h a t a t 20°; ( f l / f j c ) i s th e r e l a t i v e v i s c o s i t y o f t h e solv en tj
t o t h a t of w a te r; 20 wJ an^ / ^ t a r e d e n s i t i e s of w a te r a t ;
’ |
20° and th e s o lv e n t a t t ° , r e s p e c t i v e l y , and V i s th e p a r t i a l ■
s p e c i f i c volume of t h e p e p t i d e . j
i
The v a lu e s f o r ) >/ Q 2 0 ,w’ and/Ot> j
were c a l c u l a t e d b ase upon d a ta from a p p e n d ic e s I , I I , and I I I ,
of S vedberg and P ed ersen (1 9 4 0 ). ;
2. D e te rm in a tio n of th e m o le c u la r w eig h t j
j
In th e d e te rm in a tio n of t h e m o le c u la r w eig h t of |
t h e S A -p ep tid e m o ie ty , two d i f f e r e n t u l t r a c e n t r i f u g a t i o n e x - |
i
I
p e rim e n ts were c a r r i e d o u t: a) s e d im e n ta tio n e q u ilib riu m runsj,
i
and b) s y n t h e t i c boundary ru n s . j
a . S e d im e n ta tio n e q u ilib r iu m e x p e rim e n ts j
j
A te c h n iq u e w ith s h o r t columns of s o lu t i o n s j
j
(Van H olde and B aldw in, 1958) as recommended by Y p h an tis j
, • i
(1960) was u se d . A s ta n d a rd 12 m m aluminum c e n te r p ie c e c e l l !
61.
was u sed w ith t h e dummy c o u n te r b a la n c e c e n t e r p i e c e in t h e two
h o le AN-D r o t o r . F o u r - te n th s ml o f ,,FC-43" flu o r o c a r b o n j
!
(Beckman) was p la c e d in th e c e l l and 0.050 ml o f t h e SA- j
j
p e p tid e s o lu t i o n (0 .7 6 g m /d l in 0 .1 io n i c s t r e n g t h , pH 8 .0 , |
|
0 .1 M sodium c h l o r id e s o lu t i o n ) was la y e r e d o v e r t h e o i l . I
i
i
The r o t o r was run at. a speed o f a b o u t 20,000 r .p .m . t o develop;
I
i
c o n c e n tr a tio n g r a d ie n ts a t t h e m en iscu s and b a s e , and was j
th e n slow ed t o 6,166 r .p .m . When m a t e r i a l m ig r a tin g a c r o s s a j
f
g iv en s u r f a c e in a c e n t r i f u g a l d i r e c t i o n i s e x a c t l y b a la n c e d j
i
f
by t h e t r a n s p o r t c e n t r i p e t a l l y due t o d i f f u s i o n , e q u ilib r iu m j
i s o b ta in e d (Schachm an, 1 9 5 9 ). The d i s t r i b u t i o n was allowecL
t o r e a c h e q u ilib r iu m o v e r n ig h t a t 6 ,1 6 6 r .p .m . A l l s c h l i e r e n
o b s e r v a tio n s were made a t a c o n s ta n t p h a s e - p la te a n g le o f j
I
70° t o t h e v e r t i c a l , and e x p o su re s w ere ta k e n on p h o to g ra p h ic I ■
}
p l a t e s a f t e r e q u ilib r iu m was re a c h e d . E x p e rim e n ts were done j
|
a t 2 0 .8 5 ° . A s i m i l a r ru n , o f SO '-SO 1 d u r a t io n , was p erfo rm ed j
w ith t h e s a l i n e d i a l y s a t e i n s t e a d o f th e p e p tid e s o l u t i o n . '
i
P h o to g ra p h ic p l a t e s were m easured w ith th e Nikon two dim en- ;
i
I
i
s i o n a l c o m p a ra to r. |
b . S y n th e tic boundary e x p e rim e n ts |
T hese e x p e rim e n ts w ere c a r r i e d o u t in o r d e r j
i
t o o b ta in u l t r a c e n t r i f u g e p a t t e r n s in th e s y n t h e t i c b o u n d ary !
c e l l f o r t h e d e te r m in a tio n o f th e i n i t i a l , u n ifo rm p r o te in
62
c o n c e n t r a t i o n , r e q u ir e d in th e c a l c u l a t i o n o f th e m o le c u la r
w eig h t o f t h e S A -p e p tid e . A 4° s e c t o r , aluminum c e n t e r p i e c e ;
c e l l ( v a lv e - ty p e s y n t h e t i c boundary c e l l ) was used w ith t h e I
dummy c o u n te rb a la n c e in t h e two h o le AN-D r o t o r . F o u r - te n th s
ml o f t h e S A -p e p tid e s o lu t i o n (0 .7 6 g m /d l in 0 .1 i o n i c s tr e n g th ,
pH 8 .0 , O H M sodium c h l o r id e s o l u t i o n ) was p la c e d in t h e ‘
s e c t o r com partm ent and 0 .3 0 ml o f d i a l y s a t e in th e cup o f t h e ;
c e l l . The p ro c e d u re f o r a c c e l e r a t i n g t h e r o t o r and t a k i n g
p h o to g ra p h s was t h a t recommended by Schachman (1 9 6 5 ). The j
s c h l i e r e n o p t i c a l sy stem was used w ith t h e p h a s e - p l a t e a t an
a n g le o f 70° t o t h e v e r t i c a l . E x p erim en ts were p erfo rm ed a t
2 0 .8 5 ° , and t h e f i n a l r o t o r sp eed was 12,590 r .p .m .
c . R eading o f t h e p h o to g ra p h ic p l a t e s and
c a l c u l a t i o n s !
In g e n e r a l, th e m e a su rin g o f t h e p h o to g ra p h ic !
r e c o rd s was done in t h e same way as recommended by A lle r to n
(1 9 6 1 ). A ligm ent was checked f o r each fram e by h a v in g th e
h o r i z o n t a l c r o s s h a i r in t h e v ie w er b i s e c t th e h o r i z o n t a l
i
i
m arks in t h e r e f e r e n c e b a r s on e a c h s id e of t h e fra m e . The i
r e f e r e n c e b a r s a r i s e from h e m i - c y l i n d r i c a l h o le s in t h e dummy j
c o u n te r b a la n c e , and th e h o r i z o n t a l marks c o rre s p o n d t o z e ro
d e f l e c t i o n o f l i g h t w ith in t h e vacuum cham ber. V e r t i c a l d i s - j
r
ta n c e s o f t h e s c h li e r e n p a t t e r n from t h e s e h o r i z o n t a l m arks j
63
w ere m easu red f o r b o th t h e p e p tid e run ( d i s t a n c e s = Ys) and
t h e d i a l y s a t e run ( d i s t a n c e = Y b ). The v a lu e o f t h e co n cen
t r a t i o n g r a d i e n t (d c /d x ) a t t h e m id -p o in t (Xm) o f t h e c e l l is_
p r o p o r t i o n a l t o t h e d i f f e r e n c e b etw een t h e p e p tid e s o l u t i o n
and t h e s o lv e n t t r a c e s (Ys - Yb = A Y ). The m id -p o in t Xm i s
e q u a l t o ( a + b ) / 2 , w here a and b a r e t h e d i s t a n c e s from t h e
c e n t e r o f r o t a t i o n t o t h e m en iscu s and b a se o f t h e s o l u t i o n
( Y p h a n tis , 1 9 6 0 ).
S y n t h e t i c b o u n d ary r e c o r d s w ere a lig n e d by h a v in g t h e
h o r i z o n t a l c r o s s h a i r b i s e c t th e f l a t s e c t i o n s o f t h e p a t t e r n s
on e i t h e r s id e o f, and a d ja c e n t t o t h e b o u n d a r ie s . The b i
s e c t o r s were ta k e n as b a s e l i n e s in e v a l u a t i n g b o u n d ary a r e a s .
V e r t i c a l d i s t a n c e s (Y c o o r d i n a t e s ) o f t h e s c h l i e r e n p a t t e r
form t h e s e b a s e l i n e s w ere m easu red f o r p o i n t s (X o r d i n a t e s )
l o c a t e d a t e q u a l i n t e r v a l s th ro u g h o u t t h e b o u n d a ry .
F o r t h e c o m p u ta tio n o f t h e i n i t i a l , u n ifo rm p r o t e i n con
c e n t r a t i o n (Co) t h e a r e a b e n e a th t h e s y n t h e t i c b o u n d ary peak
was e v a l u a t e d . T h is can be done e i t h e r by means o f . a p l a n i -
m e te r g iv in g r e s u l t s in s q u a re c e n t i m e t e r s o r by t r a p e z o i d
sum m ation, w ith d a t a from l a r g e number o f e q u a l l y sp ac e d
p o i n t s . The v a lu e o b ta in e d by e i t h e r o f t h e s e two m ethods i s
th e n m u l t i p l i e d by 1/F. t o c o r r e c t f o r m a g n if ic a tio n in t h e x
64
d i r e c t i o n , and t h e r e s u l t a n t v a lu e i s p r o p o r t i o n a l t o Co
(Schachm an, 1 9 6 5 ).
The w eig h t a v e ra g e m o le c u la r w eig h t of t h e S A -p ep tid e
was c a l c u l a t e d by means o f t h e e q u a tio n :
m = RT ( d c /d x ) m
( l - V f
where M w and "V a r e th e anhydrous w e ig h t a v e ra g e m o le c u la r
w e ig h t and t h e p a r t i a l s p e c i f i c volume o f th e p r o t e i n ; R is
th e gas c o n s ta n t; 8 .3 1 4 x 10 7 e r g s /m o le /d e g r e e ; T i s t h e
a b s o lu te te m p e r a tu r e ; p i s t h e d e n s i t y of t h e s o l u t i o n ; w
i s t h e a n g u la r v e l o c i t y o f t h e c e n t r i f u g e r o t o r in r a d ia n s
p e r seco n d , ( r .p .m .) (2 fT ) /6 0 ; and Cm, and ( d c /d x ) m a r e th e
c o n c e n tr a tio n and c o n c e n tr a tio n g r a d i e n t a t t h e m id -p o in t
p o s i t i o n Xm.
The d e n s i t y of t h e S A -p ep tid e s o lu tio n a t 2 0 .8 5 ° was
c a l c u l a t e d by u s in g th e - f o r m u la :
/°-L = p + Cp ( l- V /2 ) (5)
w h ere/O i s t h e d e n s it y o f t h e s o lv e n t a t 2 0 .8 5 ° , and Cp i s
t h e p r o te in c o n c e n tr a tio n in gm/ml. In o rd e r t o c a l c u l a t e
t h e d e n s it y o f th e s o l v e n t p a t 2 0 .8 5 ° , t o th e d e n s it y v alu e
f o r w a te r a t t h a t te m p e r a tu re ( o b ta in e d from S vedberg and
P e d e rse n (1 9 4 0 ), ap p en d ix I I ) was added th e. " d e n s ity i n c r e -
ment " v a lu e f o r s odium c h l o r id e a t 0 .1 M o la r ity a t t h e same
65 |
te m p e r a tu re ( t h i s v a lu e was o b ta in e d from S v ed b erg and P e d e r- :
sen (1 9 4 0 ), a p p e n d ix I I I ) .
B i o l o g i c a l a c t i v i t y
The p o s s i b i l i t y t h a t e i t h e r th e S A - lip o p r o te in o r t h e ;
S A -p e p tid e may p o s s e s s b i o l o g i c a l a c t i v i t y was explored-, e s
p e c i a l l y w ith re g a rd t o p h o s p h a ta se a c t i v i t y .
A. A la k a lin e p h o s p h a ta s e . Technicon (1965)
T h is method i s b a se d on t h e h y d r o ly s is of t h e s u b s t r a t e
p h e n y l d iso d iu m p h o sp h a te ( p re p a re d by d i s s o l v i n g 2 .0 gm of
m onophenyl p h o sp h a te d iso d iu m (Mann R e se a rc h L a b o r a t o r y ) ,
3;7224 gm of e t h y e l e n e d i n i t r i l o t e t r a a c e t i c a c id d iso d iu m s a l t
(E a stm a n ), 3.2532 gm o f MgClg ( M a llin c k r o d t) , 0 .8 4 gm o f
sodium b i c a r b o n a te ( M a llin c k r o d t) , and 1 .5 8 gm o f sodium
c a r b o n a te anhydrous ( M a llin c k r o d t) , in 1 l i t e r o f d i s t i l l e d
w a te r and a d j u s t i n g t h e pH t o 1 0 .0 ) by a l k a l i n e p h o s p h a ta s e .
Sample and s u b s t r a t e were fe d i n t o t h e a u to m a tic T ech n ico n
system and in c u b a te d a t 37 °. L i b e r a te d p h e n o l was m easured
by r a p id c o n d e n s a tio n w ith a 0 .1 p e r c e n t 4 -a m in o a n tip y r in e
(Eastm an) s o l u t i o n , fo llo w e d by o x id a tio n w ith p o ta ssiu m
f e r r i c y a n i d e r e a g e n t ( p re p a r e d by d i s s o l v i n g 5 .0 0 0 gm of
K^Fe(CN)g. ( M a llin c k r o d t) In 1 l i t e r o f d i s t i l l e d w a te r) t o
g iv e a re d p ro d u c t w hich was m easured c o l o r i m e t r i c a l l y a t '
505mp,. A s e r i e s o f p h e n o l s ta n d a r d s (T e c h n ic o n ) r a n g in g
from 20p,g/ml t o 200|ag/m l w ere run s im u l t a n e o u s l y .
CHAPTER I I I
RESULTS
I I s o l a t i o n and C h e m ic al A n a ly s is o f S A -L ip o p ro te in
i N orm al q u in e a p i g , human and cow lu n g s h av e b een fo u n d t o
;c o n ta in 7 .5 ± 2 .5 m illig r a m s o f S A - l ip o p r o t e i n com plex p e r gram
jo f w et lu n g t i s s u e .
T a b le 1 shows t h e c h e m ic a l c o m p o sitio n o f t h e t h r e e s p e c i e s [
o f S A - l ip o p r o t e i n s i s o l a t e d in o u r L a b o r a to r y .from g u in e a p i g
(n = 4 ) , human (n = 6 ) , and cow (n = 6 ) lu n g s . I n s p e c t i o n o f t h i s
[ ta b le r e v e a l s fchat t h e t h r e e s p e c i e s o f l i p o p r o t e i n s h a v e n e a r l y
[ i d e n t i c a l c h e m ic a l c o m p o s itio n . P e p t i d e m o i e t i e s r e p r e s e n t a b o u t ;
60 p e r c e n t o f t h e w hole S A - l i p o p r o t e i n s . The l i p i d f r a c t i o n s
c o n s i s t m a in ly o f a b o u t 70 p e r c e n t p h o s p h o l i p i d s , 6 p e r c e n t ;
[ t o t a l c h o l e s t e r o l , 16 p e r c e n t t r i g l y c e r i d e s and 10 p e r c e n t '
;f a t t y a c i d s . No a tte m p t was made t o e s t i m a t e t h e am ount o f c h o - ;
[ l e s t e r o l e s t e r s , o r t o s e p a r a t e and d e te r m in e t h e com ponents o f I
;th e p h o s p h o lip id f r a c t i o n s , s i n c e t h e s e h av e b een f a i r l y w e l l j
[ e s t a b l i s h e d and r e p o r t e d in t h e l i t e r a t u r e ( P a t t l e and Thomas, ' j
1961; K la u s , e t j a l . , 1961; F i n l e y , e t a l . , 1962; Adams and
F u jiw a r a , 1963; Abrams and T a y lo r , 1964; A bram s, 1966; H a r la n ,
e t a l . , 1966; M cC lenehan, 1 9 6 7 ).
TABLE' I
CHEMICAL COMPOSITION OF VARIOUS LUNG SURFACE-ACTIVE l ipo pr o t e in sa
C o n s titu e n t
B
Guinea P ig
n
Human o
o
P e p tid e (N x 6.25) 59.2
+
0 .7 0 “ 1 60.0
+
0 .65" 59.7
+
0.59^
P h o sp h o lip id 28.2
.+
0.50 28.0
+
0 .57 2 8 .1
+
0.48
T o ta l C h o le s te ro l 3.3
+
0.10 2 .3
+
0 .16 2.5
+
0.18
T r ig ly c e r id e s 5.7
+
0.30 5 .6
+
0 .24 5.5
+
0.25
F a tty A cids 4 .6
+
0.20 4 .1
+
0 .24 4.2
+
0.24
A. C ontent in 100 m illig ra m s of d r ie d w eight m a te r ia ls
B. Average of fo u r d e te rm in a tio n s '
C. A verage of s ix d e te rm in a tio n s
D. Average of s ix d e te rm in a tio n s
* . S ta n d a rd d e v ia tio n s
! - 70
i
|D e l i p i d a t i o n
Amounts o f t o t a l c h o l e s t e r o l , p h o s p h o lip id s and t o t a l
■ n itro g en p r e s e n t b e f o r e and a f t e r d e l i p i d a t i o n o f 1 m l a l i q u o t s
;o f a c o n c e n t r a t e d s u sp e n s io n of cow lu n g S A - l ip o p r o t e i n a r e g iv e n
;in T a b le 2.
L ip o p r o t e i n m a t e r i a l s were l i p i d e x t r a c t e d a c c o r d in g t o th e
p r o c e d u r e s o f F o lc h (1 9 5 7 ), Scanu (1 9 6 6 ), and w ith a new m ethod
;d e v e lo p e d in o u r L a b o ra to r y f o r t h i s p u r p o s e . P e p t i d e r e c o v e r i e s
;w ere 9 1 .7 , and 8 J .5 p e r c e n t a f t e r one and tw o s u c c e s s i v e e x t r a c
t i o n s , r e s p e c t i v e l y , w ith t h e t e c h n iq u e o f F o lc h a t room te m p e r -,
M ature. N i n e t y - f i v e p e r c e n t p e p t i d e r e c o v e r y was o b ta in e d a f t e r
;one e x t r a c t i o n a t -1 0 ° a c c o r d in g t o th e te c h n iq u e p ro p o se d by
jScanu, and f i n a l l y 9 5 .8 p e r c e n t o f p e p tid e r e c o v e r y was o b ta in e d
jwhen t h e l i p i d e x t r a c t i o n was c a r r i e d o u t a c c o r d in g t o o u r t e c h
n iq u e a t - 7 0 ° . L o s s e s 'o f p e p tid e co u ld be p a r t i a l l y due t o m ech-
: a n i c a l h a n d lin g o f m a t e r i a l d u r in g d e l i p i d a t i o n , o r m ig h t be
'a t t r i b u t e d t o t h e p r e s e n c e of a p e p t i d e - l i p i d com plex s o lu b l e in
jth e o r g a n ic p h a s e , s i m i l a r to t h a t r e p o r t e d f o r o t h e r l i p o p r o t e i n
\
;(S can u , 1 9 6 6 ). The p r e s e n c e o f s u c h a com plex was n o t i n v e s t i
g a t e d h e r e .
]
i L i p i d a n a l y s i s c o n d u c te d on t h e d e l i p i d i z e d m a t e r i a l s have
shown t h a t t h o s e e x t r a c t e d a t room te m p e r a tu r e by F o l c h T s
TABLE 2
TOTAL CHLESTEROL, PHOSPHOLIPID AND TOTAL NITROGEN CONTENT IN 1 ML.
OF A CONCENTRATED SUSPENSION OF C O W LUNG' SA-LIPOPROTEIN BEFORE AND
. AFTER DELIPIDATION WITH THREE DIFFERENT TECHNIQUES
C ontent In C h o le s te ro l P h o sp h o lip id N itrogen
M g%
%
M g%
%
M g% %
S A -L ipoprotein 2.5 100 2 8 .1 100 9 .6 i ob
E x tra c te d Once, F o lch 1.05 42* 1.97 7* 8.8 91.7*
E x tra c te d Twice , F olch 0.47 31* 0.28 1* 8 .4 87.5*
E x tra c te d Once, Scanu N D —
1.0 3* 9 .1 95.0*
E x tra c te d ' Once, Jim enez N D
M —
ND
M M
9.2 95.8*
* P e r c e n t of t h e o r i g i n a l c o n c e n tra tio n
ND Not d e te c ta b le by th e a n a l y t i c a l methods used
te c h n iq u e s t i l l c o n ta in 31 p e r c e n t o f t h e o r i g i n a l c h o l e s t e r o l
|and 1 p e r c e n t o f t h e p h o s p h o lip id s a f t e r two s u c c e s s iv e e x t r a c
t i o n s . - On t h e o th e r h an d , 3 p e r c e n t o f t h e o r i g i n a l p h o s p h o lip id
i :
*was found in t h e d e l i p i d i z e d m a t e r i a l s a f t e r one e x t r a c t i o n a c
c o r d in g t o S c a n u ’ s te c h n iq u e , b u t no c h o l e s t e r o l was d e t e c t e d .
D e l i p i d a t i o n a t -7 0 ° removed e s s e n t i a l l y a l l c h o l e s t e r o l
|and p h o s p h o lip id s from t h e S A - lip o p r o te in m a t e r i a l s .
i
:S o l u b i l i t y o f t h e D e l i p i d i z e d S A -L ip o p ro te in
S o l u b i l i t y s t u d i e s o f t h e d e l i p i d i z e d cow lu n g S A -lip o -
;p r o t e i n m a t e r i a l s a r e sum m arized in T a b le 3. I t can be seen t h a t j
|t h e l i p i d - f r e e m a t e r i a l s a r e i n s o l u b l e in d i s t i l l e d w a te r , and
| ' ;
I in a c i d , o r n e u t r a l sodium c h l o r i d e s o l u t i o n s . They may be
| s l i g h t l y s o lu b le in 0 .1 m o la r sodium c h l o r id e s o lu t i o n a t pH -10, -
;and f a i r l y s o lu b le in 0 .1 n o rm al sodium hydroxyde s o l u t i o n s . . Sol-*
u b i l i t y v a lu e s were low a t +4° b u t c o n s id e r a b ly in c r e a s e d a t +24° ;
|(room te m p e r a t u r e ) .
■ I t wqs o b se rv e d t h a t t h e i n s o l u b l e r e s id u e s o r g e ls d i s -
:so lv e d on a d d itio n o f 6 M u r e a . I t was a l s o found t h a t t h e l i p i d r
f r e e S A -p e p tid e i s n o t d i a l y z a b l e .
TABLE 3
SOLUBILITY VALUES OF THE DELIPIDIZED C O W LUNG SA-LIPOPROTEINA
j ■ ■ ■ - , ■ , . . . . . . - - — , . , , - .
j - - = - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ' ' " ‘ ' n ■ ' - - - - - - - - - - - - - - - - - - - - - - - -
| +4° \ +24°
I
| gm % gm %
Sodium c h lo r id e , 0 ,1 M, pH 5.0
Sodium c h lo r id e , 0 .1 M, pH 7.0
Sodium c h lo r id e , 0 .1 M, pH 10.0 0.10 0.15
Sodium hydroxyde, 0 .1 N 0.50 0.85
D i s t i l l e d w ater — ,
A. C o n te n t'o f p e p tid e in s o lu tio n was e s tim a te d from i t s t o t a l n itro g e n c o n te n t
' j
IP u r i t y of S A -P ep tid e
i A n a l y t i c a l u l t r a c e n t r i f u g a t i o n , e l e c t r o p h o r e s i s and g e l
. f i l t r a t i o n were em ployed t o a s s e s s t h e p u r i t y o f th e i s o l a t e d
;S A -p e p tid e . In a d d i t i o n , e f f o r t s w ere made t o d e te rm in e w h eth er
-th e c a r b o h y d ra te s p r e s e n t were an i n t r i n s i c p a r t of t h e p e p tid e
-or r e s u l t e d from c o n ta m in a tio n w ith a g ly c o p r o te in .
j
: U l t r a c e n t r i f u g a l a n a l y s i s have shown t h e p re s e n c e of a t
l e a s t two main d i s t i n g u i s h a b l e com ponents. In a d d i t i o n , two
Ism ail and v ery r a p i d l y moving peaks c o u ld be o b se rv e d a t th e b e
g i n n i n g of th e s e d im e n ta tio n v e l o c i t y e x p e rim e n ts . T hese tw o
I
Ihigh m o le c u la r w eig h t com ponents w ere o b se rv e d in th e s c h l i e r e n
I p a t te r n s when t h e r o t o r speed was r e l a t i v e l y low , a p p ro x im a te ly
;20,000 r .p .m .
By c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s tw o main e l e c t r o p h o r -
i e t i c b o u n d a rie s were fo u n d . A l a r g e s y m m e tric a l peak ( f a s t mov
i n g ) , and a s m a ll, more slo w ly moving com ponent, c lo s e t o th e '
o r i g i n , w hich was e s tim a te d by d e n s ito m e tr y t o r e p r e s e n t l e s s
jthan 5 p e r c e n t o f t h e t o t a l p e p tid e ( F ig . 1 ) .
; G el f i l t r a t i o n on S ephadex G-100 showed t h a t a l l t h e p ro -
Ite in m a t e r i a l emerged fro m t h e column a s a s i n g l e peak ( F ig . 2 );
i
jno p r o t e i n was d e t e c t e d in l a t e r f r a c t i o n s , e i t h e r by t h e b i u r e t
i
i
r e a g e n t , o r by a b s o rb a n c e a t 280 m illim ic r o n s w a v e le n g th in t h e
s p e c tro p h o to m e te r . P r o te in r e c o v e r ie s from Sephadex G-100 were
o f t h e o r d e r of 90-95 p e r c e n t.
When t h e i s o l a t e d S A -p ep tid e was exam ined s p e c tr o p h o to -
m e t r i c a l l y o v e r t h e ran g e o f v i s i b l e and u l t r a v i o l e t l i g h t , i t
showed o n ly one a b s o rp tio n p eak , a t 280 m illim ic r o n s w av e le n g th
( F ig . 3 ).
| F i g . 1 . C e llu lo s e a c e t a t e e l e c t r o p h o r e t i c p a t t e r n o f S A -p e p tid e .
j _
j E l e c t r o p h o r e s i s : B a r b i t a l b a r b i t u r i c b u f f e r , pH 8 .6 , i o n i c
i
|s t r e n g t h 0 .0 5 , 1 .5 ma p e r s t r i p f o r t h i r t y m in u te s . S t a i n i n g :
j
jPonceau S and N i g r o s i n . S t r i p s w ere a n a ly z e d in an A n a ly tr o l
I R e c o rd in g D e n s ito m e te r I n t e g r a t o r (Beckm an).
76
FIGURE 1
F i g . 2 . B e h a v io r o f S A -p e p tid e when g e l f i l t e r e d on a S e p h a d e x '
G-100 colum n o f medium g r a i n . P e p t i d e was e l u t e d b y means o f a
0 .1 m o la r sodium c h l o r i d e s o l u t i o n , a t pH 1 0 .0 .
Absorbancy at 280 m illim icron s
FIG URE 2
1.00
.50
00
100 150 200 0 50 250
E lution volum e in m l
F i g . 3 . A b s o r p t i o n s p e c t r a o f S A -p e p tid e
80
FIGURE 3
2.00
X ! 1. 00
.00 1 j
240 280 320 360 400 440
W avelength in m illim icro n s
82
C h em ical A n a ly s is o f S A -P e p tid e
The d e l i p i d a t i o n p r o c e d u re d e v e lo p e d in o u r L a b o r a to r y
; y ie ld e d S A - l ip o p r o t e i n m a t e r i a l s a p p a r e n t l y f r e e of l i p i d s . The
i s o l u b l e and p u r i f i e d S A -p e p tid e m o ie ty h a s shown t o be c o n s t i
t u t e d o f 1 5 .7 3 ± 0 .0 5 p e r c e n t t o t a l n i t r o g e n , 1 .4 0 ± 0 .0 1 p e r
:c e n t g lu c o s a m in e , 0 .7 0 ± 0 .0 1 p e r c e n t m e th y l p e n to s e ( a s L -
j f u c o s e ) , 3 .0 0 ± 0 .0 2 p e r c e n t n e u t r a l h e x o se ( a s g a l a t o s e +
i
(M annose), an d 0 .7 5 ± 0 .0 1 p e r c e n t s i a l i c a c i d ( a s N - a c e t y l
iN eu ram in ic A c i d ) , and no d e t e c t a b l e l i p i d s . ^ b l e 4 shows a com
p a r a t i v e s tu d y o f t h e c h e m ic a l c o m p o s itio n d a t a f o r b o th SA-
r
l i p o p r o t e i n and f o r i t s S A -p e p tid e m o ie ty . As o b s e rv e d in T a b le
' A , among t h e c a r b o h y d r a t e s , n e u t r a l h e x o s e i s t h e m a jo r com ponent
|
i Q u a n t i t a t i v e , i n d i v i d u a l d e t e r m in a ti o n o f D - g a l a c t o s e o r D-
E
jm annose, o r o f any h ex o sam in e o t h e r th a n g lu c o s a m in e was n o t
ia tte m p te d .
:P e p tid e - C a r b o h y d r a t e M o iety
I As i l l u s t r a t e d in T a b le 4, s i a l i c a c i d a c c o u n te d f o r a b o u t
!
|
]0.75 p e r c e n t o f t h e w e ig h t o f t h e S A - p e p tid e . S t u d i e s w ith t h e
i
jp e p tid e s o l u t i o n s h a v e shown t h a t t h e s i a l i c a c i d c o n t e n t d i d n o t
jchange a f t e r p r e c i p i t a t i o n w ith e i t h e r 5 p e r c e n t t r i c h l o r o a c e t i c
I
i
ia c id o r 0 .6 m o la r p e r c h l o r i c a c i d (T a b le 5 ) .
TABLE '4
CHEMICAL COMPOSITION OF SA-LIPOPROTEIN AND OF SA-PEPTIDE
S A -L ipoprotein SA -Peptide
% %
P r o te in :
T o ta l N itro g en
L ip id :
P h o sp h o lip id
T o ta l C h o le s te ro l
T r ig ly c e r id e s
F a tty A cids
C arb o h y d rate:
Glucosam ine
M ethyl p e n to se (a s L -fu c o se)
N e u tra l hexose (a s galactose+m annose)
S i a l i c Acid (a s N -a c e ty l N euram inic Acid)
* S tan d ard d e v ia tio n s
100 100
9.55
+
0.08* 15.73 +
28.10
+
0.48*
2.50
+
0.18
5.50 0.25
4.20
+
0 .2 4
1.40 ± 0.01*
0.70 ± 0.01
3.00 ± 0.02
0.75 ± 0 .0 1
co
0- 1
TABLE 5
EFFECT OF VARIOUS PURIFICATION PROCEDURES
ON SIALIC ACID CONTENT OF SA-PEPTIDE
Sample A d d itio n a l P u r i f i c a t i o n Proce'dure S i a l i c Acid C ontent^
W eight %
1 None
0 .7 4
2 None
T O
T r i c h lo r o a c e tic Acid
0.75
1
0 .7 4
2 T r i c h l o r o a c e t i c Acid®
0.76
1 P e r c h lo r ic Acid*'
0 .7 3
2 P e r c h lo r ic A cid^
0.75
A. D eterm ined on sample h y d ro ly s a te s a c c o rd in g to Warren (1959)
B. P r e c i p i t a t i o n p ro ced u re w ith 5% t r i c h l o r o a c e t i c a c id
C. P r e c i p i t a t i o n p ro ced u re w ith 0 .6 M p e r c h lo r ic a c id
CO
jAmino R eid A n a ly sis I
Amino a c id s were e lu te d from th e ch ro m a to g ra p h ic columns ;
i - :
;w ith sodium c i t r a t e b u f f e r s o lu t i o n s o f i n c r e a s i n g pH (3 .2 8 and |
:4 .2 5 f o r a c i d i c and n e u t r a l am ino a c i d s , and 5 .2 8 f o r b a s i c amino :
;a c i d s ) .
i ' j
The amino a c id s p r e s e n t in th e h y d r o ly s a te s o f th e SA-. ;
: i
|p e p tid e were e lu te d from th e columns in th e f o llo w in g o r d e r :
c y s t e i c a c id , a s p a r t i c a c id , th r e o n in e , s e r i n e , g lu ta m ic a c id ,
ip r o l i n e , g ly c in e , a l a n i n e , h a l f - c y s t i n e , and v a l i n e (by means
;of a b u f f e r s o lu tio n o f pH 3 .2 8 ) , m e th io n in e , a l l o - i s o l e u c i n e , :
i s o l e u c i n e , le u c in e , (g lu c o sa m in e ), t y r o s i n e , and p h e n y la la n in e
:(b y means of a b u f f e r s o lu tio n o f pH 4 .2 5 ) . The o rd e r o f e l u t i o n !
|o f th e b a s ic amino a c id s was: h y d ro x y ly s in e , l y s i n e , h i s t i d i n e ,
^ammonia, and a r g in in e (by means of a b u f f e r s o lu t i o n of pH 5 .2 8 ) . ‘
j D ata o f d u p l i c a t e e x p erim en ts a f t e r h y d r o ly s is o f t h e SA- ;
jp e p tid e f o r p e rio d s o f 22, 46, and 70 h o u rs a t 110 t 1° i s given
;in T a b le 6. Most amino a c id s w ere c o m p le te ly r e l e a s e d a f t e r
itw enty-tw o h o u rs and w ere n o t d e s tr o y e d w ith i n c r e a s i n g h y d ro ly - !
:s is tim e . S in c e th e r e c o v e r ie s of a s p a r t i c a c i d , th r e o n in e , j
! s e r i n e , g lu ta m ic a c id , h y d ro x y ly s in e , and l y s i n e d e c re a s e d as j
1 i
i |
;th e h y d r o ly s is tim e was le n g th e n e d , t h e i n i t i a l c o n te n t o f th e s e j
I
jamino a c id s was o b ta in e d by a l i n e a r e x t r a p o l a t i o n , (S m ith , e t a l .
j 86
| • 1
j 1 9 6 4 ), t o z e r o tim e o f h y d r o l y s i s , F i g u r e 4 . The i n i t i a l c o n t e n t s
|
! of. m e th io n in e and t y r o s i n ^ . w ere c a l c u l a t e d on t h e b a s i s o f an
!a v e r a g e o f t h e r e c o v e r i e s a f t e r tw e n ty - tw o h o u r s o f h y d r o l y s i s .
[Ammonia v a l u e s i n c r e a s e d w ith l o n g e r p e r i o d s o f h y d r o l y s i s ow ing
i t o t h e d e s t r u c t i o n o f am ino a c i d s an d g lu c o s a m in e . The i n i t i a l
;c o n t e n t o f ammonia was a l s o o b t a i n e d by e x t r a p o l a t i o n t o z e r o
itim e o f h y d r o l y s i s ( F i g . 4 ) . The c o n t e n t o f h a l f - c y s t i n e was
[ e s t a b l i s h e d by a v e r a g i n g d u p l i c a t e v a lu e s fro m c y s t e i c a c i d r e
c o v e r i e s a f t e r tw e n ty - tw o h o u r s o f h y d r o l y s i s o f p e r f o r m ic a c i d
[ o x id iz e d p e p t i d e .
[ B ased on t h e r e s u l t s o u t l i n e d i n T a b le 6 , t h e f o l l o w i n g
^ c o r r e c t i o n f a c t o r s h a v e b een a p p l i e d t o am ino a c i d v a lu e s o b -
[ ta in e d a f t e r tw e n ty - tw o h o u r s o f h y d r o l y s i s o f S A - p e p tid e :
[ a s p a r t i c a c i d +5.3% , t h r e o n i n e +8.1% , s e r i n e +10.4% , g lu ta m ic
a c i d +5.0% , h y d r o x y ly s i n e +8.9% , and ammonia -1 3 .7 % .
D u rin g e a c h a n a l y s i s a s m a ll n i n h y d r i n - p o s i t i v e p eak was
[ e lu te d a t t h e same b u f f e r volum e a s t h a t r e p o r t e d f o r g lu c o s a m in e
i
(b e tw e e n l e u c i n e and t y r o s i n e ) . S in c e m ost o f t h e g lu c o s a m in e
I ’
!is d e s t r o y e d d u r in g a c i d h y d r o l y s i s , c a l c u l a t i o n o f t h e g l u c o -
jsam ine c o n t e n t o f S A - p e p tid e was n o t p o s s i b l e by t h i s m e th o d .
j The c o n t e n t o f t r y p t o p h a n in t h e i n t a c t p e p t i d e was fo u n d
t o be in t h e o r d e r o f 0 .5 8 + 0 .0 1 gram s r e s i d u e p e r 100 gram s o f
TABLE 6
AMINO ACID RECOVERIES FROM SA-PEPTIDE HYDROLYSATESA
Amino Acid
Time of H y d ro ly sis
22 H r s . 46 H rs. 70 H rs.
Average or
E x tra p o la te d Value
H alf-cystigne 4.09
— —
4.09 + 0.01*
A s p a r tic Acid 8.14 7.70 7.30 8 . 60C± 0 .3 4
/- j
T hreonine 3.33 3.09 2.71 3.60 i 0.25
S e rin e 3.26 2 .7 4 2.35 3.60°* 0.37
G lutam ic Acid 12.52 11.80 11.30 13.15c± 0.50
P r o lin e 4.53 4.60 4 .5 4 4.55 + 0 .0 1
G ly cin e 4.66 4 .6 4 ' 4.66 4.65 ± 0.02
A lan in e 4.52 4 .4 6 4.47 4.48 ± 0.02
V alin e 4.55 4.86 4.87 4.76 ± 0.19
M ethionine 2 .8 4 (2 .7 9 ) (2 .7 5 ) 2 .8 4 D ± 0 .0 3
A llo - I s o le u c in e 0.15 0.12 0.13 0 .1 3 ± 0 .0 1
I s o le u c in e 4.37 4.67 4.83 4.62 ± 0.18
L eu cin e 8.40 8.60 8.80 8.60 ± 0.10
T y ro sin e 3.10 (3 .0 8 ) (3 .0 4 ) 3.10d± 0.02
P h e n y la la n in e 4.35 4 .6 4 4.63 4 .5 4 ± 0.13
H y droxylysine ' 2.02 1.90 1 .7 4 2 .2 0 ci 0 .1 1
L y sin e 5.78 5.47 5.08 6.20c+ 0.28
H is tid in e 2.45 3.31 2.32 2.36 ± 0.02
Ammonia (NH2 ) 0.80 0.88 0.95 0.69°+ 0.02
A rg in in e 6.95 6.76 6.80 6.83 ± 0.08
TABLE 6 - Continued
A. V alues e x p re sse d as grams of amino a c id re s id u e p e r 100 grams of p e p tid e
B. Value o b ta in e d by a v e ra g in g th e r e s u l t s from two 22-hour h y d ro ly s is p e r io d s .
D eterm ined on p e rfo rm ic a c id o x id iz e d p e p tid e
C. These v alu es were o b ta in e d by e x t r a p o la tin g t o z e ro tim e of h y d ro ly s is in
o rd e r t o c o r r e c t f o r a p p a re n t d e s tr u c tio n o f amino a c id s o r, in case of amide
groups (NHg)> o^der t o c o r r e c t f o r in c re a s e d l i b e r a t i o n of ammonia from th e
d e s tr u c tio n of amino a c id s
D. These v alu es a r e av erag es of th e r e c o v e rie s a f t e r 22 hours of h y d r o ly s is ; th e
p a r e n t h e t i c a l v a lu e s a re o m itte d
* . S tan d ard d e v ia tio n s
00
C D
F i g . 4 . R e c o v e rie s a s a f u n c t io n o f tim e of h y d r o l y s i s f o r
v a r io u s amino a c id s and ammonia-, g iv e n a s grams o f r e s i d u e p e r
100 grams o f S A -p e p tid e .
89
Grams o f residue per 1 0 0 grams o f Peptide
FIG URE 4
90
G lutam ic acid
12
10
A s p a rtic acid
8
6
L ysine
4
T h reo n in e
S erine
H ydroxylysine
2
A m m onia
0
80 60 40 20 0
T im e of h y d ro ly sis in h o u rs
I 9 1
j
j
jp e p tid e , as d e te rm in e d c o l o r i m e t r i c a l l y by t h e m ethod of S p ie s
j
'and Chambers (1 9 4 9 ). In p e rfo rm in g t h e S p ies-C h am b ers r e a c t i o n ■
l o r tr y p to p h a n w ith i n t a c t S A -p e p tid e , we o b ta in e d an a p p r o p r i a t e
‘ a b s o r p tio n sp e c tru m w i t h a t r u e f i n a l s o l u t i o n .
C o m p o sitio n o f S A -P e p tid e M o ie ty
j P r e s e n t e s t i m a t e s of t h e c o m p o sitio n o f t h e S A -p e p tid e
m o ie ty a r e sum m arized in T a b le 7 . In t h e seco n d colum n, mean
v a lu e s f o r t h e amino a c id s and c a r b o h y d r a te s a r e l i s t e d , t o g e t h e r
w ith t h e i r s ta n d a r d d e v i a t i o n s . The n i t r o g e n c o n te n t o f SA-
p e p t i d e p r e p a r a t i o n s , 1 5 .7 3 ± 0 .0 5 grams p e r c e n t , was u se d t o
(derive t h e f i g u r e s i n t h e t h i r d co lu m n .
i
i
| S a l i e n t f e a t u r e s of t h e S A -p e p tid e c o m p o sitio n in c lu d e t h e
p r e s e n c e of t h e amino a c id c o n s t i t u e n t s a l l o - i s o l e u c i n e and h y e r
1
d r o x y ly s in e , w ith v a lu e s o f 1 .1 and 1 7 .2 r e s i d u e s p e r 10 9 gram s
of p e p t i d e , r e s p e c t i v e l y , an d t h e p r e s e n c e o f t h e c a r b o h y d r a te s
g lu c o sa m in e , n e u t r a l h e x o s e , fu c o s e and s i a l i c a c i d w ith v a lu e s
of 8 .7 , 1 8 .5 , 4 .8 and 2 .6 r e s i d u e s p e r 10 ^ gram s o f p e p t i d e , r e
s p e c t i v e l y .
I
' The a v e ra g e m o le c u la r w e ig h t h a s been c a l c u l a t e d on t h e ■
t a s i s o f 4 h a l f - c y s t i n e r e s i d u e s p e r m ole g iv i n g a v a lu e o f
100.,454 f o r a l l r e s i d u e s . A ls o , t h e a v e r a g e m o le c u la r w e ig h t
i
i
*
TABLE 7
COMPOSITION AND MOLECULAR WEIGHT OF SA-PEPTIDE
t
i
I t
|C o n s titu e n t
Gm. of R esidue
P e r 100 gm of
P e p tid e
N. as %
of T o ta l
N itro g en
M inimal
M o lecu lar
Weight
C a lc u la te d
M o lecu lar
Weight
Assumed
Number of
R esidues
C a lc u la te d No.
of R esid u es f o r
Average M ol. Wt.
of 100,454
H a lf -c y s tin e ^ 4.09
+
0.01* 3.57 25,000 100,000 4 4.0
|A s p a r tic A cid 8.60
+
0 .3 4 6.65 1,340 100,500 75 75.0
T hreonine 3.60
+
0.25 3.17 2,810 101,160 36 35.7
S e rin e 3.60
+
0.37 3.68 2,410 ■98,810 41 41.7
!G lutam ic Acid 13.15
+
0.50 9.10 980 99,960 102 102.5
i P r o lin e 4.55
+
0.0 1 4.17 2,130 100,110 47 47.2
!G ly cin e 4.65 0.02 7.26 1,220 100,040 82 82.3
i A lan in e 4.48
+
0.02 5.6 1 1,580 99,540 63 63.6
I V alin e 4.76
+
0.19 4.28 2,080 99,840 48 48.3
!M ethionine 2 .8 4
+
0.0 3 1.93 4,600 101,200 22 21.8
j A llo - I s o le u c in e 0 .1 3 0 .0 1 0.10 90,900 90,900 1 1 .1
1 I s o le u c in e 4.62
+
0.18 3.64 2,440 100,040- 41 41.2
|L eu cin e 8.60 ± 0.10 ' 6.77 1,300 98,800 76 77.3
;T y ro sin e 3.10
+
0,02 1.69 5,260 99,940 19 1 9 .1
;P h e n y la la n in e 4 .5 4
+
0.13 2.75 3,230 100,130 31 31.1
i H y droxylysine 2.20
+
0 .1 1 2.72 5,810 98,770 17 17.3
I L y sin e 6.20
+
0.28 8.62 2,070 99,360 48 48.5
H is tid in e 2.36
+
0.02 4.60 5,810 98,770 17 17.3
I Ammonia (NH2 ) 0.69
±
0 .0 2 B 3.61 2,460 100,860 41b 40. 8b
CD
| A rg in in e 6.83 ± 0.08 15.58 2,280 100,320 44 44 .1
I'D
i _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ...................
TABLE 7 - Continued
|
i
C o n s titu e n t
Gm. of R esidue
P er 100 gm of
P e p tid e
N. as %
of T o ta l
N itro g en
M inimal
M o lecu lar
Weight
C a lc u la te d
M olecular
Weight
Assumed
Number of
R esidues
C a lc u la te d No.
of R esidues f o r
A verage Mol. Wt.
of 100,454
n
Tryptophan 0.58 ± 0 .0 1 0.55 32,260 96,780 3 3.1
Glucosamine® 1.40 + 0 .0 1 0.77 11,490 103,410 9 8 .7
N e u tra l hexose® 3.00 ± 0.02 5,400 102,600 19 18.6
M ethyl pentose® 0.70 ± 0 .0 1 20,490 104,150 5 4.8
S i a l i c Acid® 0.75 ± 0 .0 1 0.23 38,460 115,380 3 2 .6
T o ta ls 99.33 101.05 100,454® 853 856.9
;A . Value d eterm in ed on p e rfo rm ic a c id o x id iz e d p e p tid e
|B . These v alu es a re o m itted from th e t o t a l s
|C . D eterm ined c o l o r im e t r i c a l l y on th e i n t a c t p e p tid e
|D . D eterm ined c o l o r im e t r i c a l l y
|E . A verage m o le c u la r w eight f o r a l l re s id u e s
!*. S tan d ard d e v ia tio n s
U )
U l
; 94
was c a l c u l a t e d on t h e b a s i s o f 1 a l l o - i s o l e u c i n e r e s i d u e p e r
i
j
imole g iv in g a v a lu e o f 9 1 ,2 7 2 f o r a l l r e s i d u e s , b u t t h i s v a lu e
!was n o t u sed in t h e c a l c u l a t i o n o f t h e num ber o f r e s i d u e s f o r
|a v e r a g e m o le c u la r w e ig h t s t a t e d i n t h e e i g h t h column o f T a b le 7.
; A c c u r a te d e te r m in a tio n of t h e s m a ll amount o f a l i o -
i
|i s o l e u c i n e i s d i f f i c u l t b e c a u s e o f t h e m in o r b a s e - l i n e v a r i a t i o n s
jwhen t h e u s u a l q u a n t i t y o f h y d r o ly s a t e was used ( 0 .5 ml o f sam ple
:c o n t a i n i n g t h e e q u i v a l e n t o f 0 .4 7 m illig r a m s of p e p t i d e ) . The
;a n a l y s i s of t h e a c i d i c and n e u t r a l amino a c id s was a l s o p e rfo rm e d
:w ith r e l a t i v e l y l a r g e am ounts of' h y d r o ly s a t e ( 2 .8 m illig r a m s of
p e p t i d e ) t o o b ta in a s p r e c i s e e s tim a te as p o s s i b l e o f a l l o -
!i s o l e u c i n e .
! The w e ig h t an d n i t r o g e n r e c o v e r i e s g iv e n in T a b le 7 i n d i
c a t e t h a t t h e c o m p o sitio n o f t h i s p e p tid e h a s been s a t i s f a c t o r i l y
- e s tim a te d .
In T a b le 8, t h e num ber of i o n i c g ro u p s of S A -p e p tid e i s
5
;g iv e n on t h e assu m p tio n , t h a t a s i n g l e t e r m i n a l a l p h a - c a r b o x y l
!g ro u p and a l s o a s i n g l e f r e e alp h a -a m in o group a r e p r e s e n t in
j
j t h i s p e p t i d e . The s l i g h t l y g r e a t e r number o f a n i o n i c th a n c a t i -
j
Io n ic g ro u p s may be i n a c c o rd a n c e w ith t h e a c i d i c p r o p e r t i e s o f
I
i t h i s p e p t i d e w hich s h o u ld have an i s o e l e c t r i c p o i n t o f l e s s th a n
i
jPH 7.
TABLE 8
IONIC GROUPS OF THE SA-PEPTIDE
Amino No. of Groups Amino Acid No. of Groups
i
A s p a r tic Acid 75 H ydroxylysine 17
G lutam ic A cid 102 L y sin e 48
S i a l i c Acid 3 . H is tid in e 17
T erm in al a lp h a -
carb o x y l^ 1
A rg in in e
Glucosamine®
44
9
Amide groups
181
41-
T erm in al a lp h a -
• A
ammo" 1
T o ta l a n io n ic groups ( - ) 140 T o ta l c a t i o n i c groups (+) 136
A. I t was assumed t h a t a s in g le a lp h a -c a rb o x y l group and a s in g le alpha-am ino group a re
p r e s e n t in t h i s p e p tid e
B. Assumed t o be glucosam ine, r a t h e r th an N -aceth y lg lu co sam in e, re s id u e s f o r t h i s
t a b u l a t i o n
1
96
|
IP a r t i a l S p e c i f i c Volume
| The r e l a t i v e l y c o m p le te a c c o u n tin g o f th e w e ig h t o f SA-
i
jp e p tid e h a s p e r m it t e d t h e c a l c u l a t i o n o f t h e P a r t i a l S p e c i f i c
j
jVolume (V) o f t h i s p e p t i d e . To t a k e i n t o a c c o u n t t h e f o r t y am ide
jgroups p e r m o le c u le o f S A -p e p tid e i t was assum ed t h a t t h e s e w ere
ja p p ro x im a te ly e q u a l l y d i s t r i b u t e d b etw een t h e a s p a r t i c and g lu -
I
jtam ic r e s i d u e s . The a s s ig n m e n t o f t h e am ide g ro u p s h a s no s i g
n i f i c a n t e f f e c t on c a l c u l a t i o n o f V.
j The d a t a a r e g iv e n i n T a b le 9 and le a d t o a v a lu e f o r V o f
0 .7 2 6 3 cc/gm .
j TABLE 9 i
i ;
; |
| CALCULATION OF THE PARTIAL SPECIFIC VOLUM E j
I OF THE SA-PEPTIDE FROM ITS CONSTITUENTS
1
j R esidue
Grams of R esidue
P e r 100 gm P e p tid e
Wi
S p e c if ic Volume
of R esidue
ViA
P e r cen t by Volume
of R esidue j
WiVi j
| H a lf - c y s tin e 4.09 0.63 2.577
j A s p a rtic Acid 6.31 0.60 3.786 !
Asparagine® 2.29 0.62 1.419
j T hreonine 3 .’ 60 0.70 2.520
j S e rin e 3.-60 0.63 2.268 I
G lutam ic Acid 10.51 0 .6 6 6.937
Glutamine® 2 .6 4 0.67 1.769
P r o lin e 4.55 0.76 3.458
G lycine 4.65 0 .6 4 2.976
A lan in e 4.48 0 .7 4 3.315
! V alin e 4.76 0.86 4.094
■ M ethionine 2 .8 4 0.75 2.130 !
A llo -I s o le u g in e 0.13 0.90 0.117
I s o le u c in e 4.62 0.90 4.158
L eucine 8.60 0.90 7.740 ;
i T y ro sin e 3.10 0.71 2.201 |
i P h e n y la la n in e 4.5 4 0.77 3.496 !
| H ydroxylysine 2.20 0 .7 4 C 1.628 ^ :
L y sin e 6.20 0.82 5.084 ^ ;
i H is tid in e 2.36 0.67 1.581
TABLE 9 - C ontinued
i
j
j
j R esidue
i
Grams of R esidue
P e r 100 gm P e p tid e
Wi
S p e c if ic Volume
o f R esidue
ViA
P er c e n t by Volume ;
of R esidue
WiVi
| A rg in in e 6.83 0.70 4.781
i Tryptophan 0.58 0.7 4 0.429
i G lucosam ine 1.40 0.666D 0.932
N e u tra l hexose 3.00 0.613° 1.839
i M ethyl p en to se 0.70 0.678D 0.475
S i a l i c A cid 0.75 0.584 0.438
T o ta ls 99.33 72.148 ;
; 7 = 1 WiVi / £ W i = 72.148 / 99.33 = 0.7263 cc/gm
A. These v alu es of Vi f o r th e amino a c id s a re th o se given by Cohn and E d s a ll (1949)
B. The assignm ent o f th e amide groups as a s p a ra g in y l and g lu ta m in y l i s a r b i t r a r y in
t h i s c a l c u l a t i o n . For convenience th e s e were ap p ro x im a te ly e q u a lly d i s t r i b u t e d
between a s p a r t i c and g lu tam ic re s id u e s
C. C a lc u la te d from L y sin e Vi value a c c o rd in g t o Cohn and E d s a ll (1949)
D. These v a lu e s of Vi f o r th e c a rb o h y d ra te s a re th o s e given by Gibbons (1966)
U )
03
99
i
I S e d im e n ta tio n C o n s ta n ts o f S A -P e p tid e
i
| F ig u r e 5 i s a d ra w in g o f a s c h l i e r e n p a t t e r n from a s e d i -
i
j
m e n ta tio n v e l o c i t y ru n o f S A -p e p tid e .
D a ta from a t y p i c a l s e d im e n ta tio n v e l o c i t y e x p e rim e n t have
j been p l o t t e d in F ig u r e 6 f o r t h e tw o m ain com ponents o f SA -pep-
i
| t i d e ( f a s t and slow m oving m a jo r com ponents o n l y ) . The p l o t shows
| e s s e n t i a l l y two s t r a i g h t l i n e s o v e r a b ro a d ra n g e o f t h e a b s c i s s a
I ( t i m e ) , and s e d im e n ta tio n c o e f f i c i e n t s c o u ld be c a l c u l a t e d from
!
j t h e s lo p e s o f t h e s e l i n e s . The r e s u l t s o f t h e s e e x p e rim e n ts a r e
!
jsu m m arized i n T a b le 1 0 .
! . i
! C o r r e c tio n s o f t h e o b s e rv e d s e d im e n ta tio n c o e f f i c i e n t s , i
|
|S q^ q, t o t h e s ta n d a r d s t a t e w ere made a c c o r d in g t o t h e e q u a tio n
, - — I . « » . « f e ) -
1 ~ J 2 0 j = 1 .0 0 9 ; t h e d e n s i t y o f w a te r / ° 2 0 ,w = 9*9982, and t h e
i d e n s i t y o f t h e s o lv e n t = 1 .0 0 2 2 w ere c a l c u l a t e d u s in g d a t a [
|fro m S v e d b e rg and P e d e rs e n (1 9 4 0 ), a p p e n d ic e s I , I I , and I I I .
|
j In t h e c a l c u l a t i o n o f t h e c o r r e c t e d s e d im e n ta tio n c o e f f i -
i
;c i e n t v a l u e s , it" w a s assum ed t h a t V + 0 .7 2 6 cc/gm , in t h e r e f e r -
i i
i
e n c e s o l v e n t , i s t h e same a s in t h e s o l v e n t a c t u a l l y u se d i n t h e
e x p e r im e n ts .
F o r t h e e x p e r im e n ta l d a t a p r e s e n t e d in T a b le 10, c a l c u l a
t i o n s o f S2 q w, a c c o r d in g t o e q u a tio n 3, f o r t h e slo w and f a s t j
i 9 ' i
|
m oving m a jo r com ponents o f S A -p e p tid e , le a d t o t h e r e s u l t s : J
Slow moving com ponent, S20,w
F a s t m oving com ponent, S2 q )W
100
1 .5 3 x 10“ 13 s e c . o r 1.5 3 S
3.10 x 10- - 1 -3 s e c . o r 3.10S
; F i g . 5 . S c h l i e r e n p a t t e r n fro m a t y p i c a l s e d im e n ta tio n v e l o c i t y
'r u n o f S A - p e p tid e , a f t e r f i f t y - t h r e e m in u te s a t r o t o r sp e ed o f
I'
^56,100 r .p . m . S A -p e p tid e s o l u t i o n c o n t a i n i n g 0 .7 6 d l / m l p e p t i d e
■in 0 .1 M sodium c h l o r i d e pH 8 .0 , a t 2 0 .8 5 ° . P h o to g ra p h s w ere
;t a k e n w ith a d ia p h ra g m a n g le o f 70° t o t h e v e r t i c a l . T h is d i a -
;gram c o rre s p o n d s t o a p r i n t o f th e o r i g i n a l u l t r a c e n t r i f u g e p a t
t e r n . The c r o s s h a tc h e d r e g io n s and a l l b la c k l i n e s w ould be
iw h ite on t h e o r i g i n a l p l a t e s . A ls o in t h e o r i g i n a l p l a t e w ould
:be two h o r i z o n t a l l i n e s (4 ) c o r r e s p o n d in g t o t h e im age o f t h e
■phase p l a t e b o r d e r a t t h e r e f e r e n c e h o l e s (1 and 2 ) . T h e se a r e
I u se d in t r a c i n g t h e c u r v e s and in t h e m ic ro c o m p a ra to r s in c e t h e y
j f u r n i s h a good r e f e r e n c e - f o r v e r t i c a l m e a su re m e n ts. O th e r f e a -
j t u r e s in t h e p l a t e s a r e : (3 ) a i r l a y e r ; (5 ) b a s e l i n e , h o r i z o n t a l
i
jmark in t h e a i r l a y e r ; (M) m e n is c u s , and (B) b o tto m o f t h e c e l l .
;T h is d ia g ra m showed o n ly t h e tw o main s e d im e n tin g com ponents o f
|th e p e p t i d e .
FIGURE 5
C M
O
H
CM
' 7 7 7 7/ 7/ / 7 7 7 7 7 7 / / 7 / 77 777 77 777 77 77 77 /
00
7/////////// 7 7 7/ 7//// 7////// 7 7/ 7 / 7 7 7 7
j TABLE 10
i
;
j SEDIMENTATION VELOCITY EXPERIMENT OF SA-PEPTIDE
! C a lc u la tio n of S ed im en tatio n C o e f f ic ie n ts
F = 2 .1 1 4 ; T = 2 0 .8 5 °; r o t o r speed = 56,100 r .p .m .; Cp = 0 .7 6 gm /dl
i
i A B
j Time Component
A X
cm
A X
F
■ AX_
cm
(Ax + 5.700)
^•cm
lo g x
i 37 Slow 0.6800 0.3217 6.0217 0.77972
37 F a s t 0'.8450- 0.3997 6.0997 0.78531
53 Slow 0 .7 4 .5 0 0.3524 6.0524 0.78193
| ■ 53 F a st 0.9750 0.4612 6.1612 0 .78966T
; 69 Slow 0.8100 0.3832 6.0832 0.78413
: 69 F a s t 1.1050 0.5227 6.2227 0.79397
! 85 Slow 0.8750 0.4139 6.1139 0.78632
! 85
F a st 1.2350 0.5842 6.2842 0.79825
I
i
f S = . .I--, -
dx • 2. 303 d lo g x / d t _ ( s l o p e ) (2 .3 0 3 /6 0 ) \
! W 2X d t ( 6 0 ) (w2)
( 2 r r )(6 b J lUU) 2
' 60
-
s slow = 1.50
x i o -13 =
1.50S
sf a s t = 3.04 X 10~13 = 3.04S
S ed im en tatio n C o e f f ic ie n ts c o r re c te d t o t h e s ta n d a rd s t a t e : H
O
\
TABLE 10 - Continued
x x ^ x
2 0 ,« obs * 7 h f n
s 20,WJs l O W = ( 1 .5 0 ) (0 .9 9 8 1 )(1 .0 0 9 )(1 .0 1 0 ) = 1.53S
s 20,w>f a s t = (3 .0 4 )(0 .9 9 8 1 )(1 .0 0 9 )(1 .0 1 0 ) = 3.10S
A. ■ Time in m inutes a f t e r t h e r o t o r has a t t a i n e d th e f i n a l speed of 56,100 r,
B. R e fe rs t o th e two main components o f th e S A -peptide m a te r ia l
p ,m.
H
o
F i g . 6 . P l o t o f d a t a fro m a t y p i c a l s e d im e n ta tio n v e l o c i t y e x
p e rim e n t o f S A - p e p tid e , f o r t h e c a l c u l a t i o n o f t h e s lo p e s o f t h e
l i n e s f o r e a c h one o f t h e tw o m ain com ponents (s lo w and f a s t
m oving c o m p o n e n ts). D a ta from T a b le 1 0 .
Logarithm of
106
FIGURE 6
.80000
800
600
400
200
* .79000
f a s t com ponent
800
600
400
slow com ponent
200
.78000
800
600
0 20 40 60 100 80
T im e in m in u tes
| 107
i - :
!M o le c u la r W eight o f S A -P e p tid e
I „ ; ■
i '
\ A r e p r e s e n t a t i v e s e t of d a t a from one o f t h e t h r e e s y n t h e t i c
: b o u n d ary e x p e rim e n ts p e rfo rm ed w ith S A -p e p tid e m a t e r i a l s i s p r e -
is e n te d in T a b le 11. H o r i z o n t a l and v e r t i c a l c o o r d in a te s of t h e i
i s c h l i e r e n p a t t e r n a r e g iv e n in t h e f i r s t two colum ns. D i f f e r -
e n ce s (Z) betw een Y v a lu e s f o r t h e p a t t e r n and b a s e l i n e a r e g iv en ;
|i n t h e t h i r d colum n. C a l c u l a t i o n o f t h e a r e a u n d er t h e p eak was
;done by t h e t r a p e z o i d summation m ethod, by a d d in g t h e o r d i n a t e s , , j
;Z, a t t h e x i n t e r v a l s and th e n m u l t i p l y i n g t h e sum by t h e r e
s p e c t i v e x i n t e r v a l v a lu e t o o b ta in t h e a r e a c o n t r i b u t i o n in t h e
‘f o u r t h colum n. The v a lu e o b ta in e d by summing a l l t h e a r e a s
|was th e n m u l t i p l i e d by 1/F t o c o r r e c t f o r m a g n if ic a tio n in t h e
|x d i r e c t i o n , and th e r e s u l t a n t v a lu e i s Co.
j ■
I An a v e ra g e o f t h r e e d e t e r m in a ti o n s gave t h e v a lu e Co o f ;
! j
i0 .1 7 4 s q u a re c e n t i m e t e r s .
I F ig u r e 7 i s t h e s c h l i e r e n d ia g ra m f o r a s e d im e n ta tio n e q u i - ;
jlib riu m e x p e rim e n t w ith a s h o r t column o f s o l u t i o n , in w hich t h e
■ sc h liere n p a t t e r n s o f sam ple and s o lv e n t have been su p e rim p o se d .
■ In T a b le 12 a r e p r e s e n te d t h e d a t a o b ta in e d from t h i s s e d i -
]
^m entation e q u i l i b r i u m e x p e rim e n t w ith S A -p e p tid e . In t h e f i r s t
icolumn o f t h e t a b l e t h e l o c a t i o n o f t h e r e a d in g p o i n t s on t h e i
i ' !
j j
s c h l i e r e n p a t t e r n s a r e g iv e n . The h o r i z o n t a l and v e r t i c a l j
c o o r d in a te s o f t h e s c h l i e r e n p a t t e r n s and r e f e r e n c e m arks a r e
g iv en in t h e second and t h i r d colum ns. The n e t v e r t i c a l d i s
p la c e m e n ts , a t t h e m id - p o in t Sm,[Ys - YjJ i s e q u a l t o & Y .
TABLE 11
SYNTHETIC BOUNDARY EXPERIMENT OF SA-PEPTIDE
C a lc u la tio n of A rea Under th e Peak (Co)^
F ~ 2.1 1 4 ; T - 2 0 .8 5 °; r o t o r speed - 12,590 r .p .m .; Cp = 0.76 gm/dl
1 2 3 4
I
1 2 3 4
j Xcm Ycm Zcm ^ Z 'd x t r a p . Xcm Ycm Zcm £ Z-dx t r a p .
i
| 1.45 3.400
mm —
2.12 2.390 1.010
1.50 3.395 0.005 (0 .0 2 0 )(0 ,0 5 ) 2 .1 4 2.430 0.970
! 1.55 3.390 0.010
= 0.00100
2.16 2,460 0.940
| 1.60 3.380 0.010 2.18 2.500 0.910
; 1.62 3.375 0.025 ■2.20 2.565 0.835
1 .6 4 . 3.365 0.035 2.22 2.635 0.765
1 1.66 3.355 0.045 2 .2 4 2.725 0.675
! 1.68 3.345 0.055 2.26 2.810 0.590
1.70 3.340 0.060 2.28 2.895 0.505
1.72 3.330 0.070 2.30 2.980 0.420
! 1 .7 4 3.315 0.085 2.32 3.055 0.345
1.76 3.300 0.100 2 .3 4 3.125 0.275
! 1.78 3.285 0.115 2.36 3.175 0.225
1 1.80 •3.265 0.135 ro
00
3.215 0.185
1.82 3.245 0.155 2.40 3.250 0.150
1 1 .8 4 3.215 0.185 2.42 3.275 0.125
1.86 3.180 0.220 2 .4 4 3.295 0.105
1.88 3.140 0.260 2.46 3.310 0.090
H
O
I 1.90 3.100 .0.300 2.48 3.320 0.080
U D
TABLE 11 - Continued.
! i
2 3 4 1 2 3 4 !
S Xcm Ycm Zcm Z*dx t r a p . Xcm Ycm Zcm Z*dx t r a p .
1.92 3.055 0.345 2.50 3.330 0.070
1 .9 4 3.000 0.400 2.52 3.340 0.060
! 1.96 2.935 0.465 2 .5 4 3.350 0.050
(182825)(0.02)
| 1.98 2.860 0.540 2.56 ‘ 3.360 0.040
| 2.00 2.770 0.630 2.58 3.370 0.030
= 0.36565
! 2.20 2.675 0.725 2.60 .3.375 0.025
j 2.40 2.555 0.845 2.65 3.390 0.010
(0 .0 2 7 5 )(0 .0 5 )
I 2.60 2.425 0.975 2.70 3.395 0.005
- 0.001375
2.80 2.400 1.000 2.75 ' 3.400
- -
; 2.10 2.360 1.040
i
£ ( £ Z - d x > 0.368250
i
£ ( £ Z 'd x )t r u e = 0.368250 / 2.114 = 0.1740 cm2
|
c o
= 0.1740 cm2
1
A. Area under th e peak was c a lc u la te d by th e tr a p e z o id summation method
H
H
; F i g . 7 . D iagram f o r a s h o rt-c o lu m n s e d im e n ta tio n e q u i l i b r i u m ex-!
i i
I p e r im e n t, i n w hich t h e s c h l i e r e n p a t t e r n s o f sam ple and s o lv e n t ]
• 1
I !
! h av e been s u p e rim p o s e d . S A -p e p tid e s o l u t i o n c o n t a i n i n g 0 .7 6 !
i :
i ;
1 d l/g m o f p e p t i d e i n 0 .1 M sodium c h l o r i d e s o l u t i o n pH 8 .0 , a t
2 0 '.8 5 °. P h o to g ra p h s w ere ta k e n w ith a p h a s e p l a t e a n g le a t 70°
t o t h e v e r t i c a l a f t e r e q u i l i b r i u m was r e a c h e d a t a r o t o r sp eed
; o f 6 ,1 6 6 r .p . m . (1 ) and ( 2 ) , c o rre s p o n d t o t h e l e f t and r i g h t
: r e f e r e n c e h o l e s ; (3 ) a i r l a y e r ; (4 ) h o r i z o n t a l r e f e r e n c e m ark; ;
i j
i (5 ) b a s e l i n e in t h e a i r l a y e r ; (6 ) o i l l a y e r ; (7 ) b a s e l i n e in
j t h e o i l l a y e r ; Xm, m i d - p o in t; Y^, s o lv e n t t r a c e ; Ys , sam ple
i !
I t r a c e s AY, d i f f e r e n c e b etw een Ys and Y -. a t t h e m id - p o in t Xm;
I
| a , and b , m e n isc u s and b a s e o f t h e sam ple colum n, r e s p e c t i v e l y .
I l l
f
0 0
7 7 7 7 7 // 7 7 7 7 7 ///////////////// 7 7 7 7 7 7 )
C J i
V///////////////77//////////7777,
to
H
H
ro
FIGURE 7
TABLE. 12
' SEDIMENTATION EQUILIBRIUM OF SA-PEPTIDE
C a lc u la tio n of aY a t th e M id-P oint Xm in S h o rt Columns
F = 2 .1 1 4 ; T = 2 0 .8 5 °; r o to r speed = 6,166 r .p .m .; Cp = 0 .7 6 gm /dl
I L o catio n ^
i
^cm Ycm AXcm
Axcm
x cm
AYcm ;
i
|SAMPLE RUN:
!
■
i
j L eft- r e fe re n c e b a r
B a s e lin e r e fe re n c e
; A ir-sam p le l i n e
i S ^ m p le -o il l i n e
i M id -p o in t X m
0.100
mark 3.357
1.630
1.816
1.723 2.102 1.623 0.7677 6.468 1.255 ;
SOLVENT RUN:
i L e f t re fe r e n c e b a r
B a s e lin e r e fe re n c e
| A ir-sam p le l i n e
• S a m p le -o il l i n e
M id -p o in t Xm
1.000
mark 3.344
2.524
2.728
2.626 3.290 > 1.626 0.7691 6.469 0.054
AY
Xm t r u e . =
( 1 .2 5 5 ) - ( 0 .054) = 1.201 cm
(6 .4 6 8 + 6.469) / 2 = 6.469
;A. These co rresp o n d t o p r i n t s in th e o r i g i n a l u l t r a c e n t r i f u g e p a tte r n
H
W
{ C a lc u la tio n s p r e s e n t e d on T a b le 12 l e a d t o t h e r e s u l t o f
] 1
j AY — 1*201 c e n t i m e t e r s
j ;
X = 6 .4 6 9 c e n tim e te r s
; m
I B ase upon t h e e x p e r im e n ta l d a t a so f a r c o l l e c t e d , t h e
■weight a v e ra g e m o le c u la r w e ig h t f o r t h e S A -p e p tid e was c a l c u l a t e d :
{ a c c o rd in g t o e q u a tio n 4 (fro m m a t e r i a l s and m ethods)
I M = EL___ _ .( d c /dx)m ,
; « 7 i - ~ f y 7 x m ^
S in c e R = 8 .3 1 4 x 1 0 7 e r g s / d e g r e e / m o l e ; T = 2 9 4 .0 1
1(273.16 + 2 0 .8 5 ) ; V = 0 .7 2 6 cc/g m ; - 1 .0 0 4 ( c a l c u l a t e d a c c o r d - *
jin g t o e q u a tio n 5, w ith d a t a from S v e d b e rg and P e d e rs e n (1 9 4 0 ), ■
{ap p en d ices I I and I I I ) ; w2 = 4 .1 6 5 x 1 0 5 ; ( d c / d x ) m i s p r o p o r -
t
i t i o n a l t o & Y = 1 .2 0 1 cm; Xm = 6 .4 6 9 cm, and Cm i s e q u a l t o Co =
i 2 ' ’ !
■0.1743 cm . The c a l c u l a t e d w e ig h t- a v e r a g e m o le c u la r w e ig h t i s
ith u s :
i
M _ (8 .3 1 4 x 107) (1 9 4 .0 1 )________ 1 .2 0 1 ________ _ !
j w [ l - ( 0 . 7 2 6 x 1 .0 0 4 )] (4 .1 6 5 x 1 0 5) (6 .4 6 9 ) ( 0 .1 7 4 ) j
2 .3 . x 10^ gm /m ole
‘ B i o l o g i c a l A c t i v i t y o f S A -L ip o p ro te in and o f S A -P e p tid e
: The b i o l o g i c a l a c t i v i t y of t h e S A - l ip o p r o t e i n and o f th e . i
;S A -p e p tid e m o ie ty p r e p a r a t i o n s was s t u d i e d w ith r e g a r d t o a l k a - |
jlin e p h o s p h a ta s e a c t i v i t y .
D e te r m in a tio n s w ere c a r r i e d o u t in t h e p r e s e n c e o f magnesium*
I 115
|i o n s w ith d iso d iu m p h o s p h a te as s u b s t r a t e .
j
P r e p a r a t i o n s o f S A - l ip o p r o t e i n com plex showed t r a c e s o f
I a l k a l i n e p h o s p h a ta s e a c t i v i t y , w hich h a s been e s t i m a t e d a s
ia p p r o x im a te ly 0 ,0 1 K in g -A rm stro n g U n its p e r m i l l i g r a m o f d r y -
:w e ig h t l i p o p r o t e i n . The S A -p e p tid e m o ie ty p r e p a r a t i o n s showed
ino a l k a l i n e p h o s p h a ta s e a c t i v i t y .
CHAPTER IV
DISCUSSION
i P a t t l e (1 9 6 5 ), h a s s t a t e d t h a t on w o rk in g w ith t h e lu n g
i l i n i n g " i t i s e a s y t o be l e d t o e r ro n e o u s c o n c l u s i o n s " . He
ihas e m p h a siz e d t h a t s o u r c e s o f e r r o r a r e i n h e r e n t in m ethods o f
I s tu d y in g t h e lu n g s u r f a c t a n t , and t h a t t h e r e i s c o n s i d e r a b l e
'sc o p e f o r e r r o r in t h e m eth o d s u se d f o r e x t r a c t i n g S A - l ip o p r o t e i n
jfrom lu n g t i s s u e . F o r t h a t r e a s o n , when we i n i t i a t e d t h i s i n
v e s t i g a t i o n we t r i e d t o u t i l i z e an e x t r a c t i o n p r o c e d u re w hich
^ p re v io u s ly h a s been c la im e d a s s a t i s f a c t o r y , su c h a s t h e B ondur-
ja n t and M i l l e r te c h n i q u e ( 1 9 6 2 ) . In 1967, D r. S t u a r t B o n d u ran t
'k in d ly a d v is e d us t o u t i l i z e t h e new e x t r a c t i o n t e c h n i q u e p r o
p o s e d by Abrams (1 9 6 6 ) i n s t e a d o f h i s own, s i n c e he h ad shown
- th a t t h i s new t e c h n i q u e g iv e s r e p r o d u c i b l e r e s u l t s .
The c h e m ic a l c h a r a c t e r i s t i c s o f v a r io u s s p e c i e s o f lu n g
j s u r f a c e - a c t i v e l i p o p r o t e i n s i s o l a t e d in o u r L a b o r a to r y a g r e e
i
'w ith d a t a p r e v i o u s l y r e p o r t e d by o t h e r i n v e s t i g a t o r s ( P a t t l e and
3
i '
i
116
! 117
I
!
1
[Thomas, 1961; K la u s , e t _ a l., 1961; F i n l e y , e t 1962;
i
i
!Adams and F u jiw a r a , 1963; Abrams and T a y lo r , 1964; A bram s, 1966;
;H a rla n , e t a l . , 1966; M cC lenehan, 1 9 6 7 ). I t i s a p p a r e n t t h a t
't h i s l i p o p r o t e i n i s a n o n - a r t i f a c t u a l e n t i t y , c o n s i s t e n t l y i s o
l a t e d fro m lu n g s by a v a r i e t y o f t e c h n i q u e s .
The r e s u l t a o f c h e m ic a l s tu d ie s ^ o f n o rm a l g u in e a p i g , human
iand cow lu n g S A - l i p o p r o t e i n s (T a b le 1 ) , h av e shown a r e m a rk a b ly
I
s i m i l a r i t y o f c o m p o s itio n among t h e s e t h r e e s p e c i e s o f l i p o p r o -
i
; t e i n s w h ich may r e f l e c t s i m i l a r i t y o f t h e i r f u n c t i o n s in t h e
i
jlu n g o f t h e s e mammals,
! A co m p ariso n o f t h e c h e m ic a l c o m p o s itio n o f S A - l ip o p r o t e i n
[com plex w ith o t h e r h ig h d e n s i t y l i p o p r o t e i n s , s u c h as p la sm a
t
i
[ l i p o p r o t e i n s and m i t o c h o n d r i a l s t r u c t u r a l p r o t e i n s ( S h o re , 1957;
}
!
IHanahan, 1960; C o rn w e ll an d K r u g e r, 1 9 6 1 ; G reen and F l e i s c h e r ,
i •
il9 6 3 ; L e v y , e t . a l . , 1967) h a s shown t h a t t h e p e p t i d e m o i e t i e s
i
[in a l l o f t h e s e p r o t e i n s r e p r e s e n t n e a r l y 57-60 p e r c e n t o f t h e i r
[ t o t a l d r y - w e i g h t . Of t h e i r l i p i d co m p o n en ts, p h o s p h o li p i d s r e p
r e s e n t 2 0 -2 4 p e r c e n t o f t h e d r y - w e ig h t o f p la sm a l i p o p r o t e i n s ,
Ibut c o n s t i t u t e 28 p e r c e n t o f t h e S A - l ip o p r o t e i n .d r y - w e i g h t .
!
iM oreover, w h e re a s t h e t o t a l c h o l e s t e r o l o f p la sm a l i p o p r o t e i n s
[ r e p r e s e n t 1 2 -1 7 p e r c e n t , in t h e S A - l i p o p r o t e i n , t h i s c o n s t i t u t e s
jonly 2 .6 p e r c e n t o f i t s t o t a l d r y - w e i g h t . I t i s c l e a r from
j '
[th e se f i n d i n g s t h a t a lth o u g h t h e S A - l ip o p r o t e i n may be c l a s s i f i e d
! 118
ia s a h ig h d e n s i t y l i p o p r o t e i n , i t d i f f e r s from o t h e r h ig h d e n s i t y ;
I ;
:l i p o p r o t e i n s ( i n p a r t i c u l a r from p lasm a l i p o p r o t e i n s ) w ith r e s p e c t
;t o t h e p r o p o r t i o n s o f i t s v a r io u s l i p i d f r a c t i o n s . ;
: P r e v io u s s t u d i e s h av e shown t h a t a w a te r s o l u b l e , e s s e n - ;
i t i a l l y l i p i d - f r e e p r o t e i n can be o b t a i n e d .f r o m human serum h ig h
I d e n s i t y l i p o p r o t e i n s by e x t r a c t i o n w ith c o ld e t h a n o l - e t h e r j
i
j(A v ig a n , 195 7 ; S c a n u , e t a l . , 1958; S h o re and S h o re , 1962;
: !
iS canu, 1 9 6 6 ), o r w ith o t h e r c o m b in a tio n s o f o r g a n ic s o l v e n t s
isu c h as m e th y la l- m e th a n o l (M a rg o lis and L an g d o n , 1966; L evy, e t ;
: a l . , 1 9 6 7 ). H ow ever, r e c o v e r i e s and s o l u b i l i t y p r o p e r t i e s o f
■ t h e d e l i p i d i z e d p r o d u c ts v a r i e d in e a c h s tu d y , p ro b a b ly ' b e c a u s e
;of d i f f e r e n c e s in t h e e x t r a c t i o n p r o c e d u r e s em p lo y ed . In o r d e r ;
|to o b ta in a l i p i d - f r e e p e p t i d e m o ie ty from cow lu n g S A - lip o p r o -
r ;
i j
t e i n , w h ich c o u ld b e u t i l i z e d in p h y s i c a l and c h e m ic a l c h a r a c t e r - ;
I ’
j i z a t i o n s t u d i e s , lu n g l i p o p r o t e i n s w ere s u b j e c t e d t o d e l i p i d a t i o n 1
iw ith v a r io u s t e c h n i q u e s . By means o f t h e d e l i p i d a t i o n m ethod •
^developed i n our. L a b o r a t o r y , a p e p t i d e c o n t a i n i n g no d e t e c t a b l e ;
l i p i d s , 1 5 .7 3 . p e r c e n t t o t a l n i t r o g e n , and a p p r o x im a te ly 5 .8 5 |
ip er c e n t c a r b o h y d r a te was o b t a i n e d . The y i e l d s o f S A ~ p e p tid e I
| ;
: i
' a r e c o n s id e r e d s a t i s f a c t o r y and g r e a t e r th a n t h o s e o b t a in e d w ith
i i
!
i ' ;
t h e o t h e r t e c h n i q u e s u t i l i z e d in t h i s i n v e s t i g a t i o n (T a b le 2 ) .
The l o s s e s im p lie d by t h e r e c o v e r y d a t a in T a b le 2 , o c c u r r e d ;
i
[d u rin g t h e e x t r a c t i o n p r o c e d u r e , p e rh a p s o f f a u l t y te c h n i q u e in |
: 1 1 9
i
1 :
ip e rfo rm in g th e num erous m a n ip u la tio n s in v o lv e d in t h e d e l i p i d a - !
I :
t i o n o r p e rh a p s b e c a u s e o f t h e p r e s e n c e o f a p e p t i d e - l i p i d com
p l e x s o l u b l e in t h e o r g a n i c p h a s e . Such a com plex h a s b een r e - |
f
p o r t e d t o o c c u r w ith o t h e r l i p o p r o t e i n s (S c a n u , 1 9 6 6 ) . ;
T he p r e s e n c e o f c a r b o h y d r a te m o i e t i e s in o t h e r l i p o p r o t e i n s j
i ,
h a s b een r e p o r t e d p r e v i o u s l y (S c a n u , e t a l . , 1958; A braham , e t a l . j ,
| I
jl960; M a r s h a ll a n d Kummerow, 196 2 ; A y r a u l t - J a r r i e r , e t a l . , 1963; ;
S p i r o , 1 9 6 3 ; S c a n u , 1966) b u t t h i s i s t h e f i r s t tim e t h a t a d e
t a i l e d c a r b o h y d r a t e a n a l y s i s h a s been p e rfo rm e d on t h e S A - lip o -
p r o t e i n . The c o m p le te a n a l y s i s f u r n i s h e d h e r e (T a b le 4) i n d i c a t e s ;
t h a t i t s s u g a r c o m p o s itio n i s q u a n t i t a t i v e l y d i f f e r e n t fro m t h a t
r e p o r t e d f o r o t h e r l i p o p r o t e i n s . T h u s, f o r ex am p le , i t h a s been
’ found t h a t egg y o l k l i p o p r o t e i n (A braham , 1960) c o n t a i n s 0 .6 7 p e r :
Jcent g lu c o s a m in e , 1 .3 p e r c e n t n e u t r a l h e x o s e , and 0 .3 8 p e r c e n t
s i a l i c a c i d . Human serum l i p o p r o t e i n s on t h e o t h e r h an d , h a v e ;
shown a c a r b o h y d r a te c o m p o s itio n o f 1 .5 1 p e r c e n t g lu c o s a m in e , 0 .6 0
p e r c e n t f u c o s e , 0 .8 5 n e u t r a l h e x o s e , and 0 .4 8 p e r c e n t s i a l i c
a c i d (S c a n u , 1966) . I t can b e ,o b s e r v e d t h a t th e n e u t r a l h e x o s e
c o n te n t ( 3 .0 0 p e r c e n t) o f t h e S A - l i p o p r o t e i n , r e p r e s e n t s a p p r o x i- j
m a te ly tw o - and t h r e e - f o l d , r e s p e c t i v e l y , o f t h e v a l u e s f o r t h i s
| - - - ;
c o n s t i t u e n t in e g g y o lk and human serum l i p o p r o t e i n s . M o reo v er, I
! I
uhe s i a l i c a c id c o n t e n t ( 0 .7 5 p e r c e n t ) o f t h e S A - l i p o p r o t e i n , j
I
i s a p p r o x im a te ly t h r e e an d tw o ti m e s l a r g e r th a n t h e v a lu e s f o r j
! 120 ;
jth is c o n s t i t u e n t in egg y o lk and human serum l i p o p r o t e i n s , resp ec-j
i i
t i v e l y . !
j Our s t u d i e s on p u r i t y o f t h e p e p t i d e 's o l u t i o n s have shown ;
t h a t t h e s i a l i c a c id c o n te n t d id n o t change a f t e r p r e c i p i t a t i o n I
I
i
w ith two d i f f e r e n t r e a g e n ts (T a b le 5 ) . A lth o u g h n o t c o n c lu s iv e ,
[these f in d in g s su g g e st t h a t c a rb o h y d ra te s form an i n t e g r a l p a r t j
! j
!of th e S A - p e p tid e . m o lecu le and do n o t r e s u l t from t h e p re s e n c e •
i . ' !
1 ' * " * *
'o f g ly c o p r o te in i m p u r i t i e s . !
A c c o rd in g t o W in z le r (1 9 5 8 ), l i p o p r o t e i n s m ig h t be c l a s s i f i e d
a s a l i p o i d form o f g l y c o p r o te in s . S in c e l i p i d s a r e r e a d i l y r e -
j ’ £
moved from l i p o p r o t e i n s by s o lv e n t e x t r a c t i o n , w hereas rem oval
o f t h e i r c a rb o h y d ra te m o ie tie s r e q u i r e s a c id h y d r o ly s i s , t h e term j
■ " lip o g ly c o p ro te in s ” has been p ro p o sed a s a more a p p r o p r i a t e d e sig -i
n a t i o n f o r m ost l i p o p r o t e i n s by Abraham, e t a l . (1 9 6 0 ).
| The r o l e o f th e c a rb o h y d ra te m o iety in h ig h d e n s it y l i p o - j
p r o t e i n s i s n o t y e t c l e a r ; i t may be c o n s id e re d a s c o n t r i b u t i n g ;
! 1
t o t h e s t a b i l i t y o f t h e p e p tid e backbone and p e rh a p s t o th e a n t i - ;
g e n ic s p e c i f i c i t y o f t h e m o lecu le (S can u , 1 9 6 7 ).
j . :
The amino a c id c o m p o sitio n o f t h e S A - lip o p r o te in i s re p o rte d ]
I j
f o r t h e f i r s t tim e in t h i s p a p e r . The r e s u l t s o f amino a c id !
* !
h n a l y s i s a t t e s t t h e - u n if o r m ity o f o u r S A -p e p tid e p r e p a r a t i o n s , I
■ ■ |
and t h e amino acid, d i s t r i b u t i o n i s c o n s i s t e n t w ith a c a l c u l a t e d I
i
j
m o le c u la r w e ig h t o f 100,000 b a se d on a c o n te n t o f f o u r h a l f - j
1 121
!c y s tin e r e s id u e s p e r mole of p e p tid e .
i ..
|
S a l i e n t f e a t u r e s of th e amino a c id c o m p o sitio n a re t h e p r e
s e n c e of h y d ro x y ly sin e (1 7 .3 r e s id u e s p e r mole o f p e p t i d e ) , and
'th e g e o m e tric a l isom er o f i s o l e u c i n e , a l l o - i s o l e u c i n e ( 1 .1
■ residues p e r mole o f p e p t i d e ) . H y d ro x y ly sin e i s g e n e r a lly found
!o n ly in c o lla g e n and c e r t a i n o th e r c o n n e c tiv e t i s s u e p r o t e i n s .
:The c o n te n t o f t h i s amino a c i d in th e S A -p ep tid e i s a p p ro x im a te ly
;2.2 p e r c e n t, which i s l a r g e r th a n t h a t g e n e r a lly re p o rte d f o r
i
jc o lla g e n and g e l a t i n (F ru to n and Simmonds, 1959).
I t sh o u ld be em phasized t h a t o u r i d e n t i f i c a t i o n o f a l l o -
;i s o l e u c in e and h y d ro x y ly s in e was based on c h ro m ato g rap h ic e x
p e r im e n ts , where n i n h y d r i n - p o s i t i v e peaks were c o n s i s t e n t l y
I . . - '
I e lu te d a t th e same b u f f e r volumes as th o s e re p o rte d f o r s p e c i f i c
!s u b sta n c e s by o th e r i n v e s t i g a t o r s (Moore, e t a l . , 1958; Spack-
j
jman, 1962). N e v e r th e le s s , we a g re e t h a t an a b s o lu te i d e n t i f i c a -
I •
.tio n must be based on in d e p e n d e n t c r i t e r i a such as i s o l a t i o n of
;th e -compound r e s p o n s ib le f o r th e ' peak (H am ilton and A nderson,
i1955) fo llo w e d by c h e m ic a l and p h y s ic a l c h a r a c t e r i z a t i o n , p ap er
:chrom atography, e t c e t e r a , (Z a c h a riu s and T a lle y , 1962). I f our
ifin d in g s a r e c o r r e c t , th e p re se n c e of t h e s e two amino a c id s ,
j a l l o - i s o l e u c i n e and h y d ro x y ly s in e , may be o f g r e a t im p o rtan ce in
i .
jfu tu r e i n v e s t i g a t i o n s where th e y may be u t i l i z e d as m arkers in
!
j
|s e a rc h in g f o r th e p la c e o f o r i g i n o f th e s u r f a c t a n t l i p o p r o t e i n .
122
I A com parison of t h e amino a c id co m p o sitio n o f S A -lip o p ro te in
i ;
|w ith o th e r w e ll known l i p o p r o t e i n s , such a s th e s t r u c t u r a l p ro - ;
ite in from m ito c h o n d ria (G reen and F l e i s c h e r , 1 9 63), and v a rio u s ■
;serum l i p o p r o t e i n s (S h o re , 1957; M a rg o lis and Langdon, 1966;
[Levy, _et a l . , 1967), r e v e a ls q u a l i t a t i v e and q u a n t i t a t i v e d i f - |
ife re n c e s among them . The d i f f e r e n c e s betw een th e amino a c id
; co m p o sitio n of t h e s e p r o t e i n s , c l e a r l y d e m o n stra te t h a t h ig h j
(d e n s ity l i p o p r o t e i n s (from v a rio u s s o u rc e s ) do n o t s h a re th e
jsame p e p tid e com ponent; t h e i r p e p tid e m o ie tie s a r e n o t i d e n t i c a l , ;
‘and i n t e r c o n v e r s io n s can n o t t a k e p la c e by j u s t rem oving o r a d d in g ;
; )
[ li p i d c o n s t i t u e n t s . N e v e r th e le s s , i t sh o u ld be em phasized t h a t !
ja predom inance of a c i d i c amino a c id s o v er b a s ic amino a c id s i s j
Ip re se n t in a l l th e p r o te in s s tu d ie d , in c lu d in g t h e S A -lip o p ro te in .j
i ;
i Assuming t h a t a s i n g l e t e r m i n a l a lp h a - c a rb o x y l group and j
i • <
j . . . |
:a s in g l e a lp h a-am in o group a r e p r e s e n t in t h e S A -p e p tid e , c a lc u - i
j ' i
■ latio n s have shown t h a t t h e r e i s a s l i g h t l y g r e a t e r number ( 1 .4 !
[per c e n t o f t o t a l ch arg ed g roups) o f n e g a tiv e th a n p o s i t i v e
groups in t h i s p e p tid e (T ab le 8 ). T h is r e s u l t would be a n t i c i -
I >
1 i
ipated from th e a c i d i c p r o p e r t i e s of t h i s p e p t i d e . ;
! Knowledge o f th e e n t i r e co m p o sitio n o f th e S A -lip o p ro te in [
[has p e r m itte d t h e c a l c u l a t i o n of v a rio u s p h y s ic a l c o n s ta n ts o f |
[th is s u b s ta n c e . Thus, f o r exam ple, th e p a r t i a l s p e c i f i c volume
i .
|(0 .7 2 6 cc/gm) of th e S A -p ep tid e m o iety has been c a l c u l a t e d (T ab le
j 123 !
I " '
,9 ) . M oreover, i t has been p o s s i b l e t o e s ti m a t e t h e p a r t i a l s p e -
i c i f i c volume of t h e anhydrous cow lu n g S A - l i p o p r o t e i n . This
|v a lu e , 0.852 cc/gm, i s c a l c u l a t e d in t h e same way as t h e p a r t i a l
:s p e c i f i c volume o f t h e p e p t i d e . The Vi v a lu e s f o r th e S A -lip o -
:p r o t e i n c o n s t i t u e n t s used h e r e a r e t h o s e given in T able 1, f o r
i t h e cow lu n g l i p o p r o t e i n . The Vi v a lu e f o r th e p e p t i d e c o n s t i t u - !
i :
le n t (0 .7 2 6 ) i s t h a t shown in T a b le 9, and t h e Vi v alu e s f o r t h e !
I ,
I l i p i d f r a c t i o n s ( p h o s p h o lip id 1 .0 3 1 , f r e e c h o l e s t e r o l 0 .9 4 3 ,
;c h o l e s t e r o l e s t e r s 1 .0 1 , t r i g l y c e r i d e s 1 .0 8 7 , and f a t t y a c id s
1 .087) a r e o b ta in e d from d e n s i t y d a t a giv en by Onkley (1 9 5 8 ), a s
ith e r e c i p r o c a l of t h e d e n s i t y v a lu e of e a c h l i p i d c o n s t i t u e n t .
iF.rom d a t a p ro v id e d by Abrams (1 9 6 6 ), i t i s . p o s s i b l e t o c a l c u l a t e
•the v alu e s f o r f r e e c h o l e s t e r o l and c h o l e s t e r o l e s t e r s o f th e
|S A - l ip o p r o t e i n , t h e s e a r e e s ti m a t e d as 2 .3 p e r c e n t and 0 .2 p e r
|. 1
Ic e n t, r e s p e c t i v e l y . In c a l c u l a t i n g t h e 'V v alu e f o r th e S A -lip o -
jp ro te in i t i s a l s o assumed t h a t t h e s p e c i f i c volume v a lu e f o r
jthe f a t t y a c id s i s e q u a l t o t h a t o f th e t r i g l y c e r i d e s .
i From t h e c a l c u l a t e d V v a lu e f o r cow lung S A - lip o p r o te in t h e ;
i
janhydrous d e n s i t y of t h i s l i p o p r o t e i n can be e s t i m a t e d . This !
;c a l c u l a t e d d e n s i t y v a lu e of 1.17 gm/cc i s in t h e range re g a rd e d
las c h a r a c t e r i s t i c o f h ig h d e n s i t y l i p o p r o t e i n s : 1 .0 6 - 1 .2 1
I ■ - • :
I -- . ,
1 !
j ’ i
I ‘ !
i
i 124
}
j
j(H anahan, 1960; C o rn w ell and K ru g e r, 1 9 6 1 )^ .
A w e ig h t-a v e ra g e m o le c u la r w eight o f S A -p e p tid e was d e t e r
m in e d from s e d im e n ta tio n e q u i l i b r i u m d a t a w ith s h o rt columns of
'
;s o l u t i o n s , and a v alu e o f 230,000 was o b t a i n e d . The c a l c u l a t e d
m in im al m o le c u la r w eig h t of S A -p e p tid e on th e b a s i s o f i t s chemi-
;c a l c o m p o sitio n , on t h e o t h e r hand, was 1 0 0 ,0 0 0 . The d is c re p a n c y
ibetween th e s e tw o v a lu e s may be e x p la in e d by assu m in g t h a t p o ly
m e r i z a t i o n o c c u rs in s o l u t i o n , le a d i n g t o a h i g h v a lu e f o r th e
j
iw e ig h t-a v e ra g e m o le c u la r w eig h t bu t d o e s not a f f e c t t h e m o le c u la r
:w e ig h t valu e o b ta in e d from c h e m ic a l c o m p o sitio n .
Abrams (1 9 6 6 ), h as r e p o r t e d a v a lu e of 1 9 0 ,0 0 0 -2 9 0 ,0 0 0 f o r
;th e m o le c u la r w eig h t o f S A - l i p o p r o t e i n , c a l c u l a t e d from s u rf a c e
i
I
jte n s io n m easurem ents, w hich would g iv e a m o le c u la r w e ig h t o f
;1 1 0 ,000-170,000 f o r t h e S A -p e p tid e . The m o le c u la r w e ig h t f i g u r e
Iwe o b ta in e d f o r t h e S A -p ep tid e on th e b a s i s o f i t s amino a c i d
jand c a rb o h y d ra te c o m p o sitio n , may be i n c lo s e ag reem en t w ith th e
■lower v a lu e r e p o r t e d by Abrams (1 9 6 6 ).
| W ith r e g a r d t o hom ogeneity of t h e i s o l a t e d l i p i d - f r e e
jS A -p ep tid e, u l t r a c e n t r i f u g a l and e l e c t r o p h o r e t i c d a t a have shown
j
| ^A lower d e n s i t y v a lu e would be e x p e c te d f o r t h e h y d ra te d
jbind l i p o p r o t e i n s p e c i e s . F o r exam ple, i f t h e l i p o p r o t e i n were
jto b in d 0 .3 gm. w a te r p e r gram o f l i p o p r o t e i n , a d e n s i t y n e a r
il.1 3 would be c a l c u l a t e d f o r t h e h y d r a te d s p e c i e s .
|t h a t t h e m a t e r i a l i s h e te ro g e n e o u s . The S A -peptide was re s o lv e d
i
i n t o two components by c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s , and
itwo main peaks were a l s o found in se d im e n ta tio n v e l o c i t y ex
p e r im e n ts w ith t h e a n a l y t i c a l u l t r a c e n t r i f u g e . Furtherm ore,
'se d im e n ta tio n runs have a ls o r e v e a le d a t l e a s t two m inor f a s t
Isedim enting com ponents, presum ably of v ery h ig h m o le c u la r w e ig h t.
The p r o p e r t i e s o f th e S A -p ep tid e were m arkedly in f lu e n c e d .
i
jby s o lv e n t pH. S o l u b i l i t y and u l t r a c e n t r i f u g a l d a ta i n d i c a t e d
;t h a t changes in pH of t h e medium i n f l u e n c e t h e s o l u b i l i t y of SA-
p e p t i d e . A g g reg atio n o c c u rre d a t pH 4 -6 , as ev id en ced by t h e
'fo rm atio n of a g e l which d i s s o l v e d on a d d itio n o f 6 m olar u r e a .
■At pH v alu e s h ig h e r th a n 7.0 o r l e s s th a n 4.0 t h e SA -peptide b e -
jcame more s o lu b l e . However, u l t r a c e n t r i f u g a l e x p erim en ts p e r -
I '
jformed a t pH 8 .0 have shown t h e p r e s e n c e , in t h e an aly zed p ep-
i
jtid e s o l u t i o n s , of m a t e r i a l s w ith d i f f e r e n t v e l o c i t y c o n s ta n ts ,
which su g g e sts t h a t a g g re g a tio n i s s t i l l o c c u r rin g a t t h i s pH
;value. At h ig h e r pH v a lu e s s o l u b i l i t y o f th e y p e p tid e was g r e a t l y
i n c r e a s e d , and i t ap p e a re d t o be more n e a r l y m onodisperse, as
jjudged by i t s e l u t i o n from Sephadex G-100 columns as a s in g l e
jpeak when chrom atographed a t pH 10.0 w ith 0 .1 m olar sodium
I
b h l o r i d e s o l u t i o n s . ' . .
I The o bserved dependence o f s o l u b i l i t y and ten d en cy t o
a g g re g a te of S A -p ep tid e on pH o f t h e medium su g g e sts t h a t t h e
! 126 :
j .
j f o r c e s in v o lv e d a r e e l e c t r o s t a t i c . That i s , th e s o lu tio n b e h a v io r
|o f S A -p ep tid e is' in p a r t c o n t r o ll e d by i t s n e t e l e c t r i c a l ch arg e. ;
\ 1
IThe ten d e n c y of S A -lip o p ro te in t o a g g re g a te when l i p i d s a r e r e
m oved r e c a l l s com parable b e h a v io r o f o th e r l i p o p r o t e i n s such as
i
1 human serum l i p o p r o t e i n s (Shore and Shore, 1962; Sanbar and
lA laupovic, 1963; Scanu, 1966^ m ito c h o n d ria l s t r u c t u r a l p r o te in s
i
|(Green and F l e i s c h e r , 1963), and s t r u c t u r a l p r o te in s of c e l l
imembrane system s (R ich ard so n , e t a l . , 1963; Cook, e t . a l ., 1967;
! . . . ;
iBakerman, e t .a l., 1967). T h is p o ly m e riz a tio n ten d en cy of th e
|S A -peptide, which seems t o be a g e n e r a l p h y sic o c h e m ic a l c h a ra c
t e r i s t i c o f l i p o p r o t e i n - p e p t i d e s , pro b ab ly acco u n ts f o r th e hig h
;w e ig h t-a v e ra g e m o le c u la r w eight of S A -peptide d eterm ined by s e d i-
Imentation e q u ilib r iu m e x p erim en ts. I t i s obvious t h a t t h e c a l
c u l a t e d m o le c u la r w eight v a lu e o b ta in e d on t h e b a s i s of t h e chem- ;
j i c a l com position of S A -peptide i s u n a f f e c te d by th e a g g re g a tio n
.phenomena, s in c e t h e prim ary ch em ical a n a l y s is a r e based on gram
jmasses, ra th e 'r th an m oles, of m a t e r i a l examined.
I f i t i s assumed t h a t t h e SA-peptide. c o n s is t o f a number
jof s u b - u n i t s , then t h e maximal number of t h e s e may be c a l c u l a t e d 7
Trom th e amino a c id c o m p o sitio n . S in c e i t has been found t h a t
j h a l f - c y s t i n e occurs t o th e e x te n t o f ap p ro x im a te ly fo u r re s id u e s
I j
p e r 100,000 grams o f p e p tid e , th e m inim al c h a i n .m o le c u la r w eight I
)
may be e s tim a te d a s app ro x im ately 25,000, The p re se n c e of one
127
s re s id u e of a l l o - i s o l e u c i n e and t h r e e r e s i d u e s o f try p to p h a n p e r
;100,000 grams o f p e p t i d e does n o t s e r i o u s l y c h a l l e n g e t h i s assum p
t i o n , s i n c e t h e p ro p o se d s u b u n its may n o t have i d e n t i c a l amino
;a c id c o m p o s itio n .
A model exam ple o f s u b - u n i t s t r u c t u r e i n l i p o p r o t e i n s has ;
;been r e p o r t e d p r e v i o u s l y by S h o re (1 9 5 7 ), and Shore and Shore
|( 1 9 6 2 ). I t was found (S h o re and S hore, 1962) t h a t t h e p e p tid e
component o f h ig h d e n s i t y serum l i p o p r o t e i n , which p r e v i o u s l y
h as been r e p o r t e d as h a v in g a m o le c u la r w eig h t of 9 5,000 (S h o re ,
:1957), was r e a l l y composed o f t h r e e c h a in s , e a c h one w ith an
'a p p ro x im a te m o le c u la r w eig h t o f 36,500. L a t e r , Scanu (1966)
^ rep o rted t h a t t h i s l i p o p r o t e i n s u b - u n i t was in tu r n made up of
!2S s u b u n it s w ith an a v e ra g e m o le c u la r w eight o f 2 1 ,0 0 0 ; however, ;
jhe was n o t s u re w h e th e r t h e m a t e r i a l i s o l a t e d r e p r e s e n t e d t h e
js m a lle s t s u b - u n i t of t h i s . - l i p o p r o t e i n o r some ch e m ic a l a r t i f a c t .
The c a l c u l a t e d v a lu e s o f t h e s e d im e n ta tio n c o n s ta n ts
^ s 20,w^ 1-53S and 3.10S, f o r t h e two m a jo r components o f SA-
p e p t i d e (T a b le 1 0 ) , has p e r m it t e d a c a l c u l a t i o n of t h e i r m inim al
janhydrous m o le c u la r w e ig h ts . The e q u a tio n u se d h e r e , which d e s
c r i b e s t h e r e l a t i o n s h i p betw een th e s e d im e n ta tio n r a t e and t h e
p r o p e r t i e s of t h e m o le c u le i s :
I s :: ( l - v / ? ) ( 6 5
! N f
128
jwhere s i s t h e s e d im e n ta tio n c o e f f i c i e n t , M i s t h e m in im al m ole-
jc u la r w eig h t f o r an anhydrous s p h e r i c a l s o l u t e , V i s t h e p a r t i a l
j s p e c i f i c volume of t h e s o l u t e , i s t h e d e n s i t y o f t h e s o lv e n t
I
| - r \ - i
I a t s ta n d a r d s t a t e , N i s A v o g ad ro fs number, 6.02 x 10 m o l e c u l e s /
■mole, and j 0 i s t h e f r i c t i o n a l f a c t o r which i s a f u n c t io n of t h e
isiz e o f t h e s e d im e n tin g m o le c u le s (Schachm an, --1965) .
|
| Assum ing t h a t t h e S A -p e p tid e i s an an h y d ro u s s p h e r i c a l
s o l u t e , t h e f r i c t i o n a l f a c t o r t a k e s t h e form
j o = 6 rr f [ o r (7)
where 1 Qo i s t h e v i s c o s i t y o f w a te r a t to , and r i s t h e r a d i u s
iof t h e anh y d ro u s s o l u t e m o le c u le . The r a d i u s o f t h e s o l u t e can
t h e n be c a l c u l a t e d from
i
! r = \ / ssJQty , (8)
; V 2 c i - v / ? ) ^ ;
; S in c e we know t h e v a lu e s o f f [ o = 0 .0 1 0 0 8 7 , and of f ^ o =
p ..9982 from a p p e n d ix I I o f S v ed b erg and P e d e rse n (1 9 4 0 ), we can
! _
juse o u r e x p e r im e n ta l d a t a f o r V and s t o c a l c u l a t e t h e m o le c u la r
! :
i
r a d i i and m inim al m o le c u la r w e ig h ts of b o t h ‘t h e slo w and f a s t
j
jnoving m a jo r components o f S A - p e p tid e . The c a l c u l a t e d v a lu e s a r e :
i •
] Slow moving com ponent, M = 8,600 gm/mole
i ‘
F a s t moving com ponent, M =24,800 gm/mole
| We a r e th u s l e d t o t h e f o ll o w i n g c o n s i d e r a t i o n s :
| __
129
|l . I f we assume t h a t t h e anhydrous S A -p e p tid e m o le c u le i s a
| s p h e re t h e o b serv ed s e d im e n ta ry s p e c ie s ( s2q w, 1.5 3 S , and
| 3.10S) should have m o le c u la r w e ig h ts o f n o t l e s s th a n 8,600
t
i
| and 2 4 ,8 0 0 , r e s p e c t i v e l y .
2 . I f , u n d e r t h e e x p e r im e n ta l c o n d i t i o n s in which t h e u l t r a -
r
: C e n t r i f u g a l a n a l y s i s was done a t pH 8 .0 , p o ly m e riz a tio n had
l
I
o c c u r r e d , t h e f a s t m oving component would r e p r e s e n t a t r i m e r
of t h e slow moving com ponent.
The c o n s i s t e n c y of t h e c a l c u l a t e d m o le c u la r w e ig h t o f 100,000
w ith t h e amino a cid c o m p o sitio n of t h i s p e p t i d e , seems t o
i
j . s u p p o rt th e view t h a t t h e s e two s e d im e n tin g s p e c i e s s h a re
!
; t h e same ch e m ic a l co m p o sitio n and do n o t c o n s t i t u t e d i f f e r e n t
i
i s u b s ta n c e s .
i
|3. We assume t h a t th e t r u e m o le c u la r w eig h t v a lu e s o f t h e s e
P s u b u n i t s - a r e much h i g h e r th an t h e lo w er l i m i t s g iv e n above,
| d e p e n d in g on t h e d e g re e of h y d r a tio n o f t h e p e p t i d e m o le c u le s,
I
I and t h e i r asymmetry o f shape.
I t has been found t h a t t h e l i p o p r o t e i n m a t e r i a l s e x h i b i t e d
Low -level a l k a l i n e p h o sp h a ta se a c t i v i t y ( 0 .0 1 K ing-A rm strong
U n its p e r m illig r a m of d ry -w e ig h t S A - l i p o p r o t e i n ) , a n d . t h a t t h e
S A -p e p tid e d id n o t show any p h o s p h a ta se a c t i v i t y . The f a c t t h a t
i
Only a l o w - l e v e l o f p h o s p h a ta se a c t i v i t y i s p r e s e n t i n t h e f a i r l y
130
[well p u r i f i e d S A - l i p o p r o t e i n m a t e r i a l s has been i n t e r p r e t e d as
[ in d i c a t i n g t h a t t h e main f u n c t io n o f t h i s l i p o p r o t e i n i s n o t as
’a p h o s p h a ta s e enzyme. On t h e o t h e r hand, i t was e x p e c te d t h a t
t h e S A -p e p tid e would n o t e x h i b i t a l k a l i n e p h o s p h a ta s e a c t i v i t y
[since rem oval o f t h e l i p i d s i s a p t t o d e n a tu r e t h e l i p o p r o t e i n j
i
and a b o l i s h any b i o l o g i c a l a c t i v i t y . j
r
?
S in c e t h e r e i s t h e p o s s i b i l i t y of c o n ta m in a tio n of SA- j
i
l i p o p r o t e i n w ith o t h e r s u b s ta n c e s su ch a s p h o s p h a ta s e enzymes, j
i
and w ith lu n g t r o m b o p l a s t i c l i p o p r o t e i n ( C h a r g a f f , 1 944), w hich j
!
e x h i b i t s p h o s p h a ta s e a c t i v i t y , i t rem ain s t o be seen 'w hether j
t h i s b i o l o g i c a l a c t i v i t y r e p r e s e n t s a c h a r a c t e r i s t i c of t h e SA- j
i
I
i
l i p o p r o t e i n o r a r i s e s from m inor i m p u r i t i e s . ' j
!
j
j F u r t h e r c h a r a c t e r i z a t i o n o f t h e s u r f a c t a n t l i p o p r o t e i n w i l l 'j
I
I ' !
p r o b a b ly depend on t h e d ev elo p m en t o f methods f o r d i s s o l v i n g \
!
j
'l a r g e r amounts o f i t in aqueous s o l u t i o n ; t h e use o f d e t e r g e n t s j
| j
;or b i l e s a l t s seems p r o m is in g in t h i s r e g a r d . S t u d i e s of p a r t i - j
! ■ !
I
jc u la r i n t e r e s t which p ro c e e d d i r e c t l y from t h e p r e s e n t work i n - i
i i
! ■ i
[elude: com parison o f s u r f a c t a n t a c t i v i t y o f l i p i d - f r e e p e p t i d e j
I ‘ • ‘ * I
I j
I ?
and t h e whole l i p o p r o t e i n w ith a s u r f a c e b a la n c e , e x a m in a tio n i
i i
of t h e p r o t e i n amino a c i d c o m p o sitio n in s p e c i e s o t h e r th a n t h e S
i
I
[cow, s t u d i e s o f t h e r a t e o f i n c o r p o r a t i o n o f l a b e l e d amino a c i d s -j
i n t o t h e l i p o p r o t e i n in v iv o and t h e h a l f - l i f e o f l a b e l e d l i p o -
j 1 1 1 1
I
p r o t e i n in v iv o , and s t u d i e s o f t h e e f f e c t s o f a i r p o l l u t a n t s on
131
j
Ithe p e p t i d e and l i p i d m o i e t i e s .
j
I F i n a l l y , I would l i k e t o say t h a t i t i s e v i d e n t from th e
jp re s e n t s tu d y t h a t much rem ains t o be l e a r n e d a b o u t th e n a t u r e
j
land b i o l o g i c a l r o l e of t h e S A - l i p o p r o t e i n . Are t h e r e s p e c i f i c
S A - l ip o p r o t e i n s w hich r e f l e c t p a r t i c u l a r ty p e s of p a th o g e n ic o r
( in h e r i t e d p r o c e s s e s ? What a r e t h e i r b io c h e m ic a l and p h y s ic o -
j
i ’
jchem ical p r o p e r t i e s ? What a r e t h e i r p h y s i o l o g i c a l r o le s and
i -
( b io l o g i c a l e f f e c t s ? Do t h e lu n g s u rfa c e ^ -a c tiv e l i p o p r o t e i n s from
i
I
mammals, b i r d s , r e p t i l e s , and am phibians s h a re s i m i l a r b io c h e m i
c a l and p h y s ic o c h e m ic a l p r o p e r t i e s ? These a r e p ro m is in g q u e s
t i o n s f o r f u t u r e w ork. T h is p a p e r w i l l h a v e 's e r v e d i t s p u rp o se
i _
(if i t su c c e e d s in s t i m u l a t i n g w id er i n t e r e s t in t h e s e q u e s tio n s
I ' ‘ ' ' ’
I r e la te d t o t h e lu n g s u r f a c e - a c t i v e l i p o p r o t e i n com plex.
CHAPTER V I
SUMMARY
S tu d ie s on t h e lu n g s u r f a c e - a c t i v e l i p o p r o t e i n complex
;were c a r r i e d out in o r d e r t o e s t a b l i s h , p r i m a r i l y , t h e biochem - ;
i
l i c a l and p h y s ic o c h e m ic a l c h a r a c t e r i s t i c s of i t s p e p t i d e m o ie ty .
1 -
i l t has been u n d e rta k e n in t h e hope t h a t new f i n d i n g s may e x te n d
{present know ledge on t h e n a t u r e o f t h e s u r f a c t a n t a g e n t and o f
{ lip o p r o te in s in g e n e r a l .
By means o f d i f f e r e n t i a l c e n t r i f u g a t i o n in s a l t g r a d i e n t s
{(Abrams, 1 9 6 6 ), s u r f a c e a c t i v e l i p o p r o t e i n complex was o b ta in e d i
^from f r e s h lu n g t i s s u e o f g u in ea p i g , human, and cow.
I B io c h e m ic a l a n a l y s i s of t h e t h r e e s p e c i e s of S A - lip o p r o te in s;
lhas shown t h a t t h e y a re re m a rk a b le s i m i l a r in c h e m ic a l com posi- ;
•tio n , which i s in a c c o rd w ith t h e i r homologous f u n c t i o n s .
A f t e r rem oval of l i p i d s w ith an e x t r a c t i o n te c h n i q u e d e v e l - ;
'oped by t h e a u t h o r , th e p e p t i d e o f t h e cow lu n g S A - l ip o p r o t e i n j
i i
was s u b je c te d t o an e x h a u s tiv e c h a r a c t e r i z a t i o n s tu d y . The d e lip -!
'id iz e d p e p t i d e c o n ta in e d no d e t e c t a b l e l i p i d s , 1 5 .7 3 p e r c e n t o f :
133
t o t a l n i t r o g e n , and a p p ro x im a te ly 5.85 p e r c e n t c a r b o h y d ra te .
i
^The s i a l i c a c id c o n te n t of t h e p e p t i d e d id n o t change a f t e r
{ p r e c i p i t a t i o n w ith t r i c h l o r o a c e t i c and p e r c h l o r i c a c i d r e a g e n ts ,
iwhich s u g g e s ts t h a t c a r b o h y d ra te s form an i n t e g r a l p a r t of t h e
jS A -peptide m o lecu le and do n o t r e s u l t from t h e p r e s e n c e of im-
j p u r i t i e s .
i I t was o b se rv e d t h a t t h e l i p i d - f r e e m a t e r i a l s a r e i n s o l
u b l e in d i s t i l l e d w a te r, in a c id , and in n e u t r a l p h y s i o l o g i c a l ;
s a l i n e s o l u t i o n s . They a r e s l i g h t l y s o lu b l e in 0 .1 m o lar sodium
{ c h lo rid e s o l u t i o n s a t pH 1 0 .0 , and s o lu b l e in 0 .1 norm al sodium
i
jhydroxyde s o l u t i o n s . The d e l i p i d i z e d S A -p e p tid e ~ is n o t d i a l y z - '
I - . - ■
a b l e .
• The s o lu b l e l i p i d - f r e e m a t e r i a l s were c o n c e n tr a te d by
| u l t r a f i l t r a t i o n th ro u g h D i a f l o Membranes and p u r i f i e d by g e l
I f i l t r a t i o n , in columns of Sephadex.
I t was o b se rv e d t h a t a f t e r t h e d e l i p i d i z e d m a t e r i a l b e -
icame s o lu b l e ( 0 .1 N NaQH), i t s p r o p e r t i e s were m ark ed ly in f l u e n c e d
iby s o lv e n t pH. In t h e pH ra n g e 4 -6 , where Jrh e p e p t i d e was n e a r l y :
i s o e l e c t r i c , i t formed a g e l , which c o u ld be d i s p e r s e d by 6 m olar
{urea. At pH v a lu e s h i g h e r th a n 7 .0 o r l e s s th a n 4 .0 t r a c e s of
! ' ;
{ in s o lu b le , p e rh a p s a g g r e g a te d , m a t e r i a l were o b se rv e d in t h e ;
| • . • , ' !
[ s o l u t i o n s . I t i s a p p a re n t t h a t t h e . s o l u t i o n p r o p e r t i e s o f t h i s j
jpept i d e m a t e r i a l a r e c o n t r o l l e d , a t l e a s t in p a r t , by i t s n e t ;
! 134 !
i j
c h a r g e .
I
! U l t r a c e n t r i f u g a l and e l e c t r o p h o r e t i c a l d a t a i n d i c a t e t h a t
|th e S A -p e p tid e p r e p a r a t i o n s a r e h e te r o g e n e o u s . Two bands ap p eared
^ a f t e r e l e c t r o p h o r e s i s o f S A -p e p tid e on c e l l u l o s e a c e t a t e s t r i p s :
;one m ajo r f a s t moving com ponent; and a m in o r one, c lo s e t o th e
o r i g i n , w hich c o n s t i t u t e d ab o u t 5 p e r c e n t of t h e t o t a l p e p t i d e .
'S e d im e n ta tio n p a t t e r n s in t h e a n a l y t i c a l u l t r a c e n t r i f u g e showed
two main com ponents, w ith s e d im e n ta tio n c o n s t a n t s ( s sq w) of
:1.53S and 3.10S, w ith m inim al a n h y d ro u s m o l e c u l a r w e ig h ts of ;
|8,600 and 24,800 (assu m in g s p h e r i c a l s h a p e ) , r e s p e c t i v e l y .
- -Amino a c id a n a l y s i s of S A -p e p tid e r e v e a l e d t h e p r e s e n c e
o f a l l t h e amino a c i d s n o rm a lly found i n p r o t e i n h y d r o l y s a t e s .
jln a d d i t i o n , two u n u s u a l amino a c i d c o n s t i t u e n t s were fo u n d , j
j 1
j a l l o - i s o l e u c i n e and h y d r o x y ly s in e . T hese r e l a t i v e l y uncommon j
I ;
:amino a c i d c o n s t i t u e n t s may s e r v e a s u s e f u l m a rk e rs f o r i d e n t i f y - ;
iing t h e p e p t i d e in o i e t y o f S A - l ip o p r o t e i n in f u t u r e s t u d i e s . The j
am ino a c i d c o m p o sitio n o f S A - l ip o p r o t e i n d i f f e r s q u a l i t a t i v e l y
and q u a n t i t a t i v e l y from t h o s e o f o t h e r w e l l known l i p o p r o t e i n s .
C a l c u l a t i o n s have shown t h a t t h e S A -p e p tid e c o n t a i n s more
n e g a t i v e th a n p o s i t i v e g ro u p s, in a c c o r d w ith i t s a c i d i c p r o p e r
t i e s .
i M o le c u la r w e ig h t d e t e r m i n a t i o n s w ith S A -p e p tid e h a v e led
j ~ -
|to v a lu e s o f 100,000 and 2 3 0 ,0 0 0 , on t h e b a s i s o f c h e m ic a l
| 135
com position and se d im e n ta tio n e q u ilib riu m d a ta , r e s p e c t i v e l y .
f
I The d isc re p a n c y between th e s e two v a lu e s may be ex p la in e d by
:assuming t h a t p o ly m e riz a tio n of SA -peptide occu rs in s o lu t i o n ,
le a d in g to a h ig h value f o r t h e w eig h t-av erag e m o le c u la r w eight
;determ ined by se d im e n ta tio n e q u ilib riu m .
A low l e v e l of a l k a l i n e p h o sp h ata se a c t i v i t y was d e te c te d .
lin S A - lip o p r o te in . No such enzym atic a c t i v i t y was found w ith
it.he SA -peptide m oiety. This b i o l o g i c a l a c t i v i t y may be c h a ra c
t e r i s t i c of t h e S A -lip o p ro te in but could a ls o r e s u l t from con-
Itam ination w ith d i s c r e t e lung a l k a l i n e p h o sp h a ta se s.
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This dissertation has been
microfilmed exactly as received 69-621
JIMENEZ-SAENZ, Jose Miguel, 1930-
STUDIES ON THE PULMONARY SURF ACE-ACTIVE
LIPOPROTEIN: ISOLATION AND CHARACTERIZA
TION OF ITS PEPTIDE MOIETY.
University of Southern California, Ph.D., 1968
Biochemistry
U niversity M icrofilms. Inc., A nn Arbor, M ichigan

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Creator Jiménez-Sáenz, José Miguel, 1930- (author)

Core Title Studies on the pulmonary surface-active lipoprotein: isolation and characterization of its peptide moiety

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1968-06

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)

Advisor Saunders, Paul R. (committee chair), Allerton, Samuel E. (committee member), Chew, Robert M. (committee member), Dunn, Arnold S. (committee member), Savage, Jay Mathers (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-635257

Unique identifier UC11361310

Identifier 6900621.pdf (filename),usctheses-c18-635257 (legacy record id)

Legacy Identifier 6900621.pdf

Dmrecord 635257

Document Type Dissertation

Rights Jimenez-Saenz, Jose Miguel

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...

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