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Studies On The Cell-Free Synthesis Of Collagen On Polysomes From Chick Embryo Connective Tissues

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Studies On The Cell-Free Synthesis Of Collagen On Polysomes From Chick Embryo Connective Tissues

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Content INFORMATION t o u s e r s
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Filmed
Xarox University
3 00 North Zm U Road
A"" Amor, MtcM ean ahO*
! 75-1083
TRACT, TH om s Wolfgang, 1943-
STUDIES O N T H E CELL-FREE SYNTHESIS O F C O L L A G E N
O N P O L Y SO M E S F R O M C H IC K E M B R Y O C O N N E C T IV E
j TISSUES.
U niversity o f Southern C a lifo rn ia , Ph.D., 1974
j Chewistry, b io lo g ic a l
Xerox University Microfilms f Ann Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
STUDIES ON THE CELL-FR EE SYNTHESIS OF COLLAGEN
ON POLYSOMES FROM CHICK EMBRYO CONNECTIVE TISSUES
by
Thomas Wolfgang Traut
A D isserta tio n P resen ted to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a rtia l F u lfillm ent of the
R equirem ents for the D egree
DOCTOR OF PHILOSOPHY
(C ellular and M olecular Biology)
Septem ber 1974
UNIVERSITY O F SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY SARK
LO S ANGELES, CALIFORNIA S 0 0 0 7
This dissertation, written by
..T feo aaa w . Q l .t g f l n g X x a w i t ................
under the direction of Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by The Graduate
School, in partial fulfillment of requirements of
the degree of
D O C T O R O F P H IL O S O P H Y
DISSERTATION COMMITTEE
DEDICATION
To Karyn, for sharing m y venture, and to Kevin and Jason,
b ecau se they are in terested and ask questions.
ACKNOWLEDGEMENT
The author w ish es to thank Dr. John Petruska for the
opportunity to work with him , and for h is guidance and
objective c riticism .
F or tech n ical a ssista n ce , the author is indebted to
Dr. Bernard Abbott, Dr. Sam uel A llerton, Dr. Robert Baker,
Dr. Arnold Dunn, Dr. M ichael Schneir, Dr. Harold Slavkin,
Dr. Bernard Strehler, and to Dr. Paul Benya for h is generous
gift of collagen ase.
Special thanks go to Dr. Herm an Cheung for m any, m any
hours of d iscu ssio n and co-operation , and to Dr. Tony Mark
for stim ulating c r itic ism .
ABSTRACT j
P oly so m es w ere prepared from skin, and from the wings and j
i
le g s , of 11 1/2 day chick em bryos. P olysom es from both sou rces |
w ere equally effective in stim ulating protein syn th esis in a c e ll- j
free system in the p resen ce of hom ologous preparations of j
initiation fa cto rs, and soluble factors from c e ll sap. The two
sy stem s w ere found to differ significan tly in their respon se to !
such factor preparations, and concentrations optim al for protein
syn th esis w ere determ ined for each sy stem .
By the u se of detergen ts, a large polysom e fraction was isolated ;
intact from the m icrosom al m em branes of chick skin. T hese
p olysom es had a sedim entation constant of 1600 S on lin ear su cro se
density gradients, and w ere estim ated to contain about 116 j
j
rib osom es per mRNA. C ollagen constituted over 50% of the
proteins synth esized on th ese la rge p o ly so m es, and the tim e
required to com plete one round of tran slation was determ ined to be
+ !
13. 5 - 1. 5 m inutes. I
I
The c e ll-fr e e system was able to sy n th esize and r ele a se j
protein for at le a st 40 m inutes under standard conditions, and
a n alysis of the product on acrylam ide g els dem onstrated that the |
iv I
collagen syn th esized by th is sytem included C L \ chains, C L 2 chains,
and Q chains. E xperim ents w here re-in itia tio n of ribosom es on
the mRNA was blocked, and w here the translation product was
puTse labelled after two thirds of the translation tim e had elapsed
showed that no significan t amount of C L \ chains was synth esized
while a substantial amount of C L 2 chains was form ed. The
1
| com bined evidence from th ese studies indicated that the large
l
j polysom e fraction from chick em bryo skin contained
j
1 p olycistron ic collagen m essen g er RNA.
TABLE OF CONTENTS
Page
ACKNOW LEDGEM ENT........................................................................ iii
A B S T R A C T ............................................................................. iv
LIST OF TABLES v iii
LIST OF F I G U R E S ............................................................ ix
CHAPTER
L IN T R O D U C T IO N ............................................................1
G eneral
P rocollagen
Synthesis of Collagen
Studies of C ollagen Synthesis on
P o ly so m es
H. MATERIALS AND METHODS 14
P reparation of M aterials and Solutions
P reparation of T issu es from Chick
Em bryos
P reparation of Whole P o ly so m es
P reparation of Soluble Enzym es
P reparation of Initiation F actors
A ssa y s of Enzym e Solutions and P oly so m es
F ractionation of P o ly so m es on Sucrose
G radients
Incubation of T issu e S lices
Incubation System for P rotein Synthesis
A ssay for the Incorporation of ^H P rolin e
A ssa y for T ranslation T im e
C ollagenase A ssa y of N ewly Synthesized
P rotein s
C ollagenase A ssa y of P o lysom e F raction s
Polyacrylam id e Gel E lectrop h oresis
D eterm ination of Sedim entation Values
E stim ation of the Number of R ibosom es
per P olysom e.
D eterm ination of the T ranslation T im e
CHAPTER
ID RESULTS AND DISCUSSION .
Size of CoUagen P o ly so m es
Studies on P rotein Synthesis in the
C e ll-F r e e System .
Studies on C ollagen Synthesis with
Isolated P olysom e F raction s.
IV SUMMARY
BIBLIOGRAPHY
Page
35
100
106
vii
LIST OF TABLES
Table Page
1. S Values for F raction s of 15-60% Sucrose
Gradient . . . . . . 31
2. P olysom e D istribution on Sucrose Gradientt 46
3. D istribution of R ibosom es on D ifferent
P o ly so m es . . . . . . 48
4. Dependancy of the C e ll-F r e e System on
Various F actors . . . . . 76
5. C ollagenase Studies on Newly Synthesized
P rotein s . . . . . . 81
6. T ranslation Tim e of P oly so m es . . 93
7. T ranslation R ates for D ifferent M olecules 95
8. A n alysis of P u lse-L a b elled C ollagen on
A crylam ide G els . . . . 97
v iii
LIST OF FIGURES
Figure Page
1 . C alibration of Sucrose G radients . . 22
2 . Sedim entation P r o files of L abelled P o ly so m es
from Chick Em bryo Connective T issu es 36
3 . Sedim entation P ro file of P o ly so m es from a
N on-deter gent H om ogenate of Chick Skin 39
4. Sedim entation P ro file of P o ly so m es from a
D etergent Hom ogenate of Chick Skin . 41
5. Sedim entation P ro file of P o ly so m es from a
D etergent Hom ogenate of Chick Skin . 43
6. The E ffect of Mg’ * " * " on P rotein Synthesis
with P o ly so m es from Chick Skin . . 52
7. The E ffect of Mg+" * ’ on P rotein Synthesis
with P o ly so m es from Chick Wings and L egs 54
8. The E ffect of K+ on P rotein Synthesis with
P o ly so m es from Chick Skin . . . 56
9. The E ffect of K* on P rotein Synthesis with
P o ly so m es from Chick Wings and L egs 58
10. The E ffect o f pH 5 E nzym es on P rotein
Synthesis with P o ly so m es from Chick Skin 64
11. The E ffect of pH 5 E nzym es on P rotein
Synthesis with P o ly so m es from Chick Wings
and L e g s ................................................................... 66
ix
Figure
12.
13.
14.
15.
16.
17.
18.
19.
20.
Page
The E ffect of AS7 0 E nzym es on P rotein
Synthesis with P o ly so m es from
Chick Skin . . . . . . 68
The E ffect of AS7 0 E nzym es on P rotein
Synthesis with P o ly so m es from Chick
Wings and L egs . . . . . 70
The E ffect o f Initiation F actors on P rotein
Synthesis with P o ly so m es from Chick
Wings and L egs . . . . . 72
The E ffect of Initiation F a cto rs on P rotein
Synthesis with P o ly so m es from Chick Skin 74
A bsorbance Spectrum of Isolated P olysom e
F raction s . . . . . . . 79
C ollagenase T reatm ent of Heavy P o lysom es 84
The Synthesis and R elea se of P rotein s on
P o ly so m es from Chick Skin . . . 87
T ran slation T im e of the Medium P olysom e
F raction P repared from Chick Skin . 89
T ranslation T im e of the Heavy P olysom e
F raction P repared from Chick Skin . 91
x
CHAPTER I
INTRODUCTION
G eneral
Studies on the syn th esis of collagen focus on a m olecu le that is
i
i
a lm ost ubiquitous throughout the anim al kingdom , produced in som e j
i
v a riety in a com plicated sequence of m etabolic step s, and
functional in a d iv ersity of extra cellu lar environm ents throughout
!
the body. C ollagen is found in all verteb rate and m ost
l
invertebrate organ ism s down to the sim p le P o rifera (1). C ollagen i
is the m ajor structural elem en t of the connective tissu e s of which
i
bone, ca rtila g e, and skin a re the m o st notable. Once
!
incorporated into a fib ril, the collagen m olecu le is v ery stab le,
I
though it can read ily be d isso lv ed by collagen ase enzym es in c a se s
such as the developing m orphogenesis of tadpoles (2). C ollagen
can a lso be u tilized as an energy r e se r v e and b ecom es catabolized
under conditions of str e s s (3). I
i
Due to its com plexity, studies describing collagen abound with
l
!
a v a riety o f term s used to identify th is m olecu le at different sta g es |
i
of its syn th esis or deploym ent. The term 'co lla g en 1 is u sed to |
designate the m olecu le in any, or a ll, of its form s; 'procollagen* |
. - - 1
r e fe r s to a p recu rso r form of collagen as it is syn th esised within
the c ell; 'tropocollagen* in d icates an extracellu lar collagen
m o lecu le, which is com posed of th ree individual C L chains aligned
in a trip le h elix conform ation. T ropocollagen m o lecu les, with a
m olecu lar weight of 3 00,000 daltons, becom e integrated into
m icrofilam en ts and la rg er m icro fib rils arranged in a uniform ,
repeating pattern (4). The tropocollagen m olecu le has a s t if f ,
ro d -lik e structure w hose stab ility is la rg ely due to non-covalent
bonding betw een its th ree aligned C L chains (5). This m olecu le is
| |
h elica l throughout m o st of its length of 1000 A except for sm all
I
a m in o-term in al and carboxy term in al portions (6, 7).
C onsiderable p ro g ress has been m ade in studies of the collagen i
C L chains, and peptide fragm ents of the two m ajor species,C L j
and C L 2 have been sequenced in m any lab oratories (8-12) and the :
j ;
seq uen ces of the CL chains have now been constructed. A
com parison of C L chains from m any tissu e s ind icates a fair amount j
j
of evolutionary hom ology (13). I
i |
A n alysis of peptides produced by cyanogen brom ide cleavage
! I
in d icates the ex isten ce of a v a riety of CL chains with sligh tly '
different am ino acid com position s. The prevalent form of collagen |
I
I
found in skin and bone has the com position CL}^ CL2(14), but sin ce j
variant form s of CL j have been found, th is CL chain of skin and
bone is now designated C L j(I). An CLi(II) collagen has been
2
found as the m ajor sp e cie s in cartilage (15), and the
tropocollagen has the com position (Cli(H))3 (16,17). An C ll( t it )
has recen tly been found in human skin (18), and CCi(jy) in b a se
m ent m em branes (19). The finding of th ree different C ll chains
in a sin gle tissu e (19) reflec ts a greater amount of genetic
inform ation coding for collagen , and su ggests the need for control
m echanism s at the le v e l of transcrip tion or tran slation to regulate
this genetic ex p ressio n into the d esired product.
P rocollagen
i B ecause of the difficulty of form ing trip le stranded collagen
l
in vitro with denatured Cl chains, Speakman (20) proposed that
; collagen be syn th esized as a la rg er p recu rsor m olecu le having an
I
additional 'reg istra tio n peptide1 at the am ino term inal end of each
Cl chain to fa cilita te alignm ent and linking of 3 Cl chains into a
trip le h elica l procollagen m o lecu le. Speakman suggested that
once th is procollagen m o lecu le was form ed, ex cisio n of th ese
additional term inal peptides would convert procollagen to collagen.
Imbued with this sudden raison d 'etre, procollagen was
im m ed iately d iscovered (21-23), and ch aracterized as having
| Cl chains with a m olecu lar weight 115, 000 - 125,000 in skin and
j
i cartilage (24) and 140, 000 in basem ent m em brane (25, 26). As
; reported by F e s sle r et a l. (27) the alignm ent and linking of the
C i chains into a trip le h elix is probably facilitated by disulfide
b rid g es, sin ce they d isco v ered that the 'reg istra tio n peptide'
contains cystein e resid u es which are not found in the h elica l region
of the m o lecu le. Concom ittant with the d isco v ery of procollagen,
L apiere and colleagu es reported the ex isten ce of a procollagen
peptidase a ctivity in bovine skin (28) and the subsequent
purification of this enzym e (29).
Synthesis of C ollagen
The syn th esis of collagen has been review ed by a number of j
I authors (24, 30-33) and v ery com p rehen sively treated in a recen t 1
| :
review (34). Although this p r o c ess has been predom inantly
j !
j studied in fib rob lasts and chondroblasts in situ , collagen syn th esis
! has a lso been dem onstrated in a v a riety of n on -fib roblast cell !
I i
cultures (35) and in spinal cord epithelium (36). !
A. Intracellular E vents 1
i
i
The b io g en esis of collagen begins with the syn th esis of
| pro-CL chains on p olyrib osom es that are predom inantly found
|
I attached to the m em branes of the endoplasm ic reticu lum (37).
| !
! I
C ertain proline and ly sin e resid u es of the pro-CL chain are
| I
; hydroxylated eith er on the nascent polypeptide chain (38), or on the j
j
r elea sed chain (39) by th eir resp ectiv e h yd roxylases. It is quite
I
, reasonable that hydroxylation o ccu rs both during and after protein |
4 ;
L i
syn th esis (40). Although hydroxylation is not required, per se .
for the syn th esis and r e le a se of a pro-CL chain (41), hydroxylation
is n e c e ssa r y for the trip le h elix conform ation that endows collagen
with its structural rigid ity (42). Studies with p rolyl hydroxylase
have also shown that the rate of hydroxylation in c r ea se s as the
! substrate a ssu m es the h elica l conform ation (43). To
I
J recapitulate th is v e ry im portant sequence: hydroxylation begins
| on the nascen t pro-CL chains and continues on the com pleted
I
I
j pro-CL chains as th ey begin to align and b ecom e linked via
{ disulfide bridges in the amino term in al peptide; when som e
I
m inim um , but n e c essa ry , degree of hydroxylation has been
achieved the three chains a ssu m e a trip le helix conform ation,
i
after which hydroxylation m ay be com pleted. D uring, or after,
| the hydroxylation sequence certain of the hydroxylysine resid u es
I
are glycosylated .
The glycosylating enzym es a re m em brane-bound (33) w hile
j the hydroxylating enzym es have b een reported a s being soluble
! (33) or as being attached to the m em branes of the endoplasm ic
i
reticulum in studies using ferritin -la b elled antibodies (37,44).
This discrepancy m ay be reso lv ed by the proposition that the
enzym es a re solu b le, but form a com plex with the nascent
p rocollagen on the m em brane-bound p o lysom e, and are th erefore
I
seen as m em brane-bound under the electro n m icro sco p e after
being tagged with the fe r ritin lab elled antibodies.
In order to fu lfill its functional ro le, the com pleted
p rocollagen m u st be extruded from the c e ll, but th ere is s till
considerab le con troversy over the m echanism of extrusion.
In an electron m icro sco p ic study of ca rtilage s lic e s (45) evidence
is p resen ted that collagen could be d irectly extruded into the
ex tra cellu lar m atrix through openings in the cistern ae of the
endoplasm ic reticu lum . H ow ever, in an excellen t study by
| W einstock and Leblond (46) follow ing the path of lab elled
j i
procollagen in odontoblasts, it is shown quite c lea r ly that
I :
! i
j procollagen tra v els via G olgi vacu oles into sec r eto r y v e s ic le s .
As the sec r eto r y v e s ic le s a re extruded through the cell m em brane j
i
I !
| they bu rst, and the p rocollagen appears to becom e converted to <
I !
| collagen . A fter studying the syn th esis of pro collagen in the
l
p resen ce of inhibitors of m icrotub ules and m icro fila m en ts, !
i
i
E hrlich and B orn stein (23) concluded that the tra n scellu la r m ove- j
I m ent of procollagen from the endoplasm ic reticu lum was
i
I fa cilita ted by m icrotu b u les.
!
The con version of p rocollagen to collagen o ccu rs either
|
during the extrusion of p rocollagen through the c e ll m em brane,
i
or in the extern al m edium . P rocollagen has been iso la ted out
sid e the c e ll in cultured fib rob lasts (47) and in rat skin (48), |
! !
indicating that p rocollagen peptidase m ay e x ist in the ex tra - |
cellu lar environm ent, although it has been claim ed that th is sam e
enzym e is located within the c e ll in studies on cartilage (23). It
m ay be that this enzym e has a cellu la r locu s for its action, and
not a ll of the procollagen m o lecu les a re efficien tly cleaved to
collagen - th is has been dem onstrated in cattle and sheep
afflicted with derm atosporaxis (49). A ltern atively, th ere m ay
be rea l d ifferen ces in the location of this enzym e that a re tis s u e -
sp ecific or sp e c ie s -s p e c ific .
i
I
1 B. E xtracellu lar Events
| The N -term in al ly sin e s of collagen a re converted to aid e-
l
hydes (allysin es) v ia oxidative deam ination by ly sin e oxid ase.
: Through an aldol condensation in tram olecu lar bonds are form ed
betw een two a lly sin es of adjacent chains of a collagen m olecu le
j (50,51). In a som ewhat sim ila r fashion, in term olecu lar
I
c r o ss-lin k s are m ade via the reaction of an a lly sine with a ly sin e
on a second collagen m o lecu le to form a Schiff b a se, which upon
reduction form s a stable lysin on orleu cin e c r o ss-lin k (52-55).
While intram olecular c r o ss-lin k s have been found only in the
am ino term inal region , in term olecu lar c r o ss-lin k s have been
dem onstrated betw een the am ino term in al region o f one collagen
i
m o lecu le and the car boxy term inal region of another, in a
quarter stagger overlap (56). T hese c r o ss-lin k s confer greater
stab ility on the individual collagen m o lecu le, and on the aggregates
that lead to the form ation of fib r ils.
The form ation of in solu ble collagen fib ers from soluble
I
collagen m o lecu les has been studied exten siv ely (57-60), and is
related to the amount and type of m u cop olysacch arid es and
glycoproteins that in teract with collagen in the extracellu lar
m atrix of connective tis s u e s (60-63), although th ere does not seem I
j
to be any interdependance or control in the syn th esis of collagen I
and m u cop olysacch arid es (64). The in teraction s of th ese
! I
1 i
I m o lecu les in the form ation of fib rils has been studied ex ten sively
! j
by Wood (65-70), and a ll the m ucop olysaccharides tested w ere
S
| effective in causing soluble collagen to p recip itate and form
fib r ils, though the ra tes of fib ril form ation w ere found to vary.
: i
I i
: It has been su ggested by m any of th ese authors that the wide
|
v a riety of patterns ob served in collagen fib ril form ation in j
different tis s u e s m ay be a unique function o f the type and amount
i
i
| of m u cop olysaccharid es that a re availab le in the extracellu lar
| m atrix of the different tis s u e s . An illu stra tio n of th is d iv ersity
can be found in the e y e, w here collagen is the m ajor structural j
l
I
component of the tough, opaque s c le r a and of the clea r, j
■ t
; j
transparent cornea.
Studies of C ollagen Synthesis on P olysom es
E fforts to elucidate the nature of collagen syn th esis at the
ribosom al le v e l began in the laboratory of A lexander Rich and his
co llea g u es. They analyzed p olysom es from chick em bryos
labelled with radioactive proline on lin ear su cro se gradients and
found the la b el a sso cia ted with a v a riety of p o ly so m es,
including the h ea v iest fraction in the p ellet (71, 72). Sim ilar
stud ies by Fernandez-M adrid a lso showed a wide distribution
of polysom al m a teria l ranging in s iz e up to 2000 S, a variety of
which carried nascen t peptides lab elled with proline (73). But
while th ese studies on chick em bryos dem onstrated the
ex isten ce o f v ery large p o ly so m es, G oldberg and G reen (74)
showed that p olysom es from cultured fib rob lasts w ere rather
sm a ll, with a peak at 180 S, and no polysom es la rg er than
I
330 S.
To r e so lv e th is con troversy o ver the s iz e of collagen polysom es|
Speakman, in a th eo retica l paper (75), speculated that a j
collagen polysom e for a sin gle Cl chain would have 30 to 35
rib osom es (which is reasonable sin ce the la rg er p ro -C l chain j
i
had yet to be d iscovered ). D ism issin g the chick em bryo |
studies due to im p lied technical p rob lem s, Speakman adopted
the sedim entation valu e reported by Goldberg and G reen,
concluding that th is polysom e with 30 to 35 rib osom es should j
9
have a s iz e of about 320 S. Since liv e r p olysom es with only 10
rib osom es have a sedim entation constant of 320 S (76), the value
proposed by Speakman was unfortunately, and unreasonably, low.
P o ssib ly influenced by Speakman's paper, studies on
i
collagen polysom es reported s iz e s of 300-400 S in the next few
y ea rs (77-79). Perhaps th is value was not questioned because
j a study on calf len s p olysom es reported la r g e polysom es that
| w ere e a sily d isp ersed with tryp sin (80), indicating that
!
i la rg e p olysom es m ight be artifactu al a g g regates. In review ing
i this field , Fernandez-M adrid concluded that collagen polysom es
|
had a sedim entation constant of about 450 S, and p o ssib ly larger
i
(31). H ow ever, sin ce som e of the above stu d ies (74, 77-79)
used som ewhat shallow er su c ro se gradients and long cen tri-
I
fugation p erio d s, it would have been im p o ssib le to detect v ery
la rge p olysom es on the gradient elution p ro file sin ce th ese large
J p olysom es would have been p elleted on the bottom of the
j centrifuge tube.D iegelm an et a l. (37) have recen tly reported a
|
rapidly sedim enting m em brane-bound p olysom e com plex from
em bryonic chick tib iae.
!
i
To m easu re the rate of collagen sy n th esis e a rlier studies
follow ed the appearance of lab elled collagen outside the c e ll, and
have reported syn th esis tim es in cartilage of one m inute (39) and
3. 3 m inutes (81). Such valu es are difficult to interp ret sin ce
th ere a re num erous step s with p o ssib le erro rs in th is approach,
and th is is best illu stra ted by a reported value of 60 m inutes
for the appearance of la b elled collagen from basem ent m em brane
(25). Much m ore relia b le valu es have been obtained by Vuust
and P ie z . T hese authors thoroughly la b elled chick em bryo
calvaria in vitro over a period of 24 hours with 3h prolin e,
and then w ere able to m ea su re the sp e cific activity of a 1 4 c
proline p u lse in the different peptides derived from collagen.
By extrapolating from valu es for the appearance of lab el at
different tim e points after addition of the p u lse, they determ ined
a syn th esis tim e for collagen CL chains of about 4 .8 m inutes (82).
A fa irly sim ila r tim e of 4. 5 m inutes has been reported for
QL 1 (II) cka * n8 in cartilage by e sse n tia lly the sam e procedure
(83). Follow ing the d isco v ery of p rocollagen , Vuust and P ie z
repeated th eir study and determ ined that pro - C L chains are
syn th esized in about 5. 8 m in utes (84). Although th ese kinetic
stu d ies have been c riticize d (47), the valu es reported for the
syn th esis tim e of collagen are quite reasonab le and m ight be
im proved only by the u se of a c e ll-fr e e sy stem for collagen
sy n th esis.
T here have been only a few stu d ies with c e ll-fr e e sy stem s
for the syn th esis of collagen . The e a r lie s t attem pt used
m ature rat skin and produced little m eaningful data b ecau se of
im m en se tech n ical d ifficu lties (85). L azarid es and Lukens
dem onstrated a low le v e l of collagen syn th esis on p olysom es
from em bryonic chicken w ings and le g s (77). K erw ar et al.
(78, 79) achieved m uch better resu lts with p olysom es from
cultured fib ro b la sts. They dem onstrated that about 15% of the
in vitro sy n th esised protein was collagen , and they w ere able to
identify this product by colum n chrom atography and acrylam ide
gel e lectro p h o resis. M ore recen tly, collagen mRNA from chick
aalvaria has been tran slated in a heterologous sy stem from I
j
Krebs II a sc ite s tum or c e lls , producing CL and Q collagen s (86).
i
This d isserta tio n p resen ts the resu lts of studies on the j
syn th esis o f collagen on p olysom es in a c e ll-fr e e sy stem . To
|
obtain a m ore efficien t sy stem for the syn th esis of collagen , j
p olysom es w ere analyzed on su c ro se gradients with the objective
l
of iso la tin g a fraction containing m o stly collagen p oly so m es. At j
the tim e that th ese stud ies w ere initiated, it was reported that a
control m echanism for tran slation of m e sse n g e r RNA m ight
involve protein factors sp ecific to the tissu e producing the mRNA
(87); th erefore a ll protein syn th esis studies w ere perform ed in j
|
a hom ologous sy stem to a ssu re the in clu sion of any n e c e ssa r y j
t
tissu e sp e cific protein fa cto rs. Although the m ajor stu d ies ,
reported h ere w ere perform ed with p olysom es from chick em bryo
skin, an analogous sy stem was prepared from wings and leg s
and used for com parative stu d ies.
CHAPTER II
MATERIALS AND METHODS
P reparation of M aterials and Solutions
A ll ch em icals w ere reagent grade, purchased from Sigma
C hem ical Co. , St. L ouis, Mo. or from C albiochem , Los
A n geles, C alifornia. U ltrapure su cro se for grad ien ts, and
ultrapure am m onium sulfate for enzym e preparations w ere
purchased from Schw artz/M ann, O rangeburg, New York.
A ll g lassw are and instrum ents w ere ste r iliz e d by baking
overnight at 280°C . P la stic v ia ls and centrifuge tubes w ere
ste rilize d with a solution of diethylpyrocarbonate at 3 m l/L (88).
Buffer solutions included sodium heparin at 500 u g /m l as an
inhibitor of RNAse activity (89, 90).
Preparation of T issu es from Chick Em bryos
F ertilized White Leghorn chicken eggs (Shaver S tarcross
strain) w ere purchased from D em ler F a rm s, Anaheim ,
C alifornia. Eggs w ere m aintained in incubators at 38°C , and
w ere norm ally used at 11 1/2 days after fertiliza tio n . This age
was a com prom ise betw een two opposing fa cto rs. It was
d esired to u se em bryos as old a s p o ssib le to m axim ise the yield
of connective tis s u e s , but it was a lso n e c e ssa r y to u se the em bryos
before the on set of the sy n th esis of keratin or fea th ers. In
studies on the syn th esis of chick em bryo collagen it has been
shown that collagen syn th esis in the skin rem ains at a high le v el
from days 8 to 14 (91) or days 13 to 16 (92). K eratin syn th esis
begins at about day 13, reaching a peak by day 16 (93). Thus
chick em bryos w ere routinely used at 11 1 /2 days after
fertiliza tio n .
A fter rem oval from the sh e ll, the em bryo was blotted dry
! with a paper tow el. An in cisio n was m ade circu m scrib in g the
| dorsal su rface, and a sm all strip of filter paper was p r e sse d to
j the skin which adhered to the paper and could be p eeled off.
I
| Strips of skin w ere co llected in ic e cold H om ogenizing Buffer
(20 mM T ris-H C l, pH 7. 4 at 4°C , 240 mM KC1; 20 mM
MgCl2 ; 120 mM su cro se; 500 u g /m l heparin). After the skin
was rem oved, the wings and le g s w ere cut off and placed into a
sep arate collectin g v e s s e l with the sam e H om ogenizing Buffer.
The tissu e s w ere p elleted by centrifugation at 5000 x g
i
for 10 m inutes on a Sorvall refrig era ted centrifuge (Ivan
Sorvall I n c ., Norwalk, C onn.) and resuspended in 2 volum es
of H om ogenizing Buffer and hom ogenized with 6 strok es of a
tight p e stle in a g la ss Dounce hom ogen izer. H om ogenization of
wings and le g s required two p relim in ary strok es with a lo o se
p e stle . The hom ogenate was centrifuged at 12, 000 x g for
15-20 m in utes, the supernate was rem oved, and the p ellet
was resuspended in 2 volum es of Hom ogenizing Buffer
containing sodium deoxycholate, 0. 5%,and Triton X-100, 0. 5%
to solu b ilize m em brane bound polysom es (90). The tissu e was
again hom ogenized with 6 strok es and centrifuged at 1 2 , 0 0 0 x g
for 15-20 m in utes. The two supernate fraction s w ere either
stored in liquid nitrogen, or used d irectly for the preparation
of polysom e or tissu e fa cto rs.
j
P reparation of Whole P olysom es
Supernates from the hom ogenized tissu e s w ere centrifuged j
in an SW 27 rotor on a Beckm an L 2-65B ultracentrifuge
I
(Beckm an Instrum ents Inc. , F u llerton, California) at 27,000
rpm for 150 m in utes. The supernate was decanted and the
i
t
I
p ellet gently rinsed tw ice with 1 m l of Incubation Buffer j
(20 mM T ris-H C l, pH 7 .4 at 37°C; 150 mM KC1; 7. 5 mM |
MgCl2 ; 6 mM m ercaptoethanol or 1 mM dithiothreitol). P ellets
w ere then resuspended in the sam e buffer by gentle stirrin g
j
or hom ogenization with a lo o se fitting teflon p e stle . The i
!
polysom e su sp en sion s appeared grey and turbid, and w ere
cla rified by centrifugation at 5000 x g for 10 m inutes. |
I
i
16 :
P o lysom e solutions w ere then dialyzed against the sam e buffer
overnight, and concentrated with Sephadex (P harm acia, U ppsala,
Sweden). This was accom plished by covering the d ia ly sis bag
with dry Sephadex powder to absorb w ater from within the
d ia ly sis bag. A ll solutions w ere stored in sm all aliquots in
liquid nitrogen. P olysom e solutions with an A2 6 0 /A 2 8 O le s s
than 1. 6 w ere considered as too contam inated and w ere not used
for protein syn th esis stu d ies.
P reparation of Soluble Enzym es
Soluble enzym es for protein syn th esis w ere prepared from
1 non-detergent p o st-m icro so m a l supernates by two different
j m ethods. A pH 5 fraction was prepared by adjusting the
supernate to a pH of 5. 2 with a cetic acid (94). A fter 30 m inutes
the precip itate was co llected by centrifugation at 15,000 x g for
20 m in utes. The precip itate was d isso lv ed in Enzym e Buffer
(20 mM T ris-H C l, pH 7 .4 at 37°C; 1 mM DTT; 0.1 mM EDTA)
and p recip itated again at pH 5. 2. The p recip itate was collected
as above, d isso lv ed again in the sam e buffer against which it was
dialyzed overnight, and stored in liquid nitrogen after being
concentrated with Sephadex.
Soluble enzym es w ere a lso prepared follow ing the
i
j m ethod of Arlinghaus et a l. (95). N eutralized protam ine sulfate
17
(10 m g /m l) was added to a final concentration of 0. 34 m g /m l
in the non-detergent supernate. This m ixture was stirred for
30 m inutes on ic e , and the p recip itate was rem oved by
centrifugation at 10, 000 x g for 20 m in u tes. The supernate was
adjusted to 100 mM T ris-H C l with 2 M T ris-H C l (pH 7. 5 at
23°C ), and to a pH of 6. 5 with a cetic acid. The solution was
then brought to 40% of saturation with am m onium sulfate by the
slow addition of 22. 6 g/100 m l w hile being constantly stirred at
4°C . A fter 30 m inutes the precip itate was rem oved by
centrifugation at 12, 000 x g for 20 m in u tes. The supernate wap
then brought to 70% of saturation with am m onium sulfar e
slow addition of 21. 0 g/100 m l of the origin al pH 6. 5 solution.
A fter 30 m inutes the precip itate was co llected as above and
d isso lv ed in Enzym e Buffer. The preparation was then dialyzed,
concentrated, and stored as d escrib ed p reviou sly. This enzym e
preparation was lab elled AS^q (for Am m onium Sulfate, 70%).
P reparation of Initiation F actors
P rotein factors required for the initiation of protein
sy n th esis w ere prepared after the m ethod of M iller and Schweet
(96). P o lysom e solutions (at concentrations of 50-100 A 26o/m l)
w ere adjusted to a fin al KC1 concentration of 0. 5 M w hile being
stir re d gently for 30 m inutes in the cold. At th is sa lt
concentration initiation factors w ere d isso cia ted from the
rib o so m es. The rib osom es w ere p elleted out of the solution
by centrifugation on the Beckm an Model L centrifuge at
10Q 000 x g for 150 m inutes with an SW 39 rotor. The upper
90% of the supernate w as rem oved and prepared as the other
enzym e fra ctio n s, and stored in liquid nitrogen.
A ssa y s of Enzym e Solutions and P olysom es
The protein concentration of the different enzym e solutions
I
was determ ined by the m ethod of Lowry (97) using bovine seru m j
i
i
album in (BSA) as a standard. The concentration of ribosom es
in a polysom e solution was determ ined by the absorbance at
260 nm , using the relationship that 1 m g /m l of rib osom es =
l
12 A2 6 O (98, 99). In m ost ca ses the concentration of polysom es j
i
was recorded as the number of optical density units at 260 nm.
I
F ractionation of P o ly so m es on Sucrose G radients
L inear su cro se density gradients w ere prepared after the j
i
m ethod of M artin and A m es (100) as m odified by Noll (76).
A gradient m aker was fashioned out of lu c ite, containing two
cylind rical w e lls, each with a volum e of 60 m l. The two w ells
w ere connected v ia a flow v a lv e, and one w ell (the m ixing w ell) |
i
1
had th ree outlet tubes at its b ase which w ere connected via I
i
a Buchler p o lystaltic pump to th ree centrifuge tu bes. To
prepare a gradient, a solution with the su cro se concentration !
I
t
19 ,
d e sir e d at the top of the gradient was placed in the fir s t w ell,
and a solution with the su cro se concentration d esired at the
bottom of the gradient was placed in the m ixing w ell, where
m ixing was accom plished by in sertion of a sonicating stir re r .
The gradient was form ed by starting the m ix er, the pump and
opening the connecting valve betw een the two w ells. To avoid
air bubbles in the connecting v a lv e, it was alw ays filled with the
top su cro se solution in advance. The su cro se solution was
primped from the m ixing w ell down the in sid e of the centrifuge
tubes. The gradient was th erefore form ed from bottom to top.
Since the two starting su cro se solutions varied considerably
in density, th ere was a difference in the atm ospheric p ressu re
controlling the flow of the ligh ter solution into the heavier one.
Thus som e adjustm ents had to be m ade in the volum es and
concentrations of the two starting solutions to achieve a lin ear
den sity gradient o f the d esired range. T hese values w ere
calcu lated as follow s
Vt = volum e of top su cro se solution
Vb = volum e of bottom su cro se solution
d{ = density of top su cro se solution
db = density of bottom su cro se solution
Vg = volum e of total gradient (all 3 centrifuge tubes).
Vg' = Vg + Vr (Vr = resid u al volum e in cham ber).
20
The solutions in the two w ells w ere balanced when
Vt dt = Vb db
The follow ing relationship s w ere derived (where C = concen
tration of su crose):
T h erefore, to m ake th ree 15-60% su cro se gradients, each with
a volum e o f 34 m l, 56. 5 m l of a 14. 54% su cro se solution was
placed in the fir st w ell and 46. 5 m l of a 60% su cro se solution
was placed in the m ixing w ell. A ll su cro se solutions w ere m ade
in H om ogenizing Buffer. Figure 1 shows the calibration curve
of two su cro se gradients that w ere co llected in 1 m l fraction s
i
and m easured on a Bausch and Lomb refractom eter (Baush I
|
and Lomb, G lendale, C alifornia). It can be seen that the j
gradients w ere v e ry n early lin ear over the entire range.
Although the top two m l of the gradient deviated slig h tly , it was
assu m ed that when an aqueous sam ple was placed on top of the
J
gradient som e dilution occu rred and thereby low ered the |
concentration to the d esired value.
I
I
To co llect fraction s from a gradient, the centrifuge tube j
w as placed in a p la stic holder containing a threaded p iercin g j
i
needle in the bottom . Once the centrifuge tube was p rop erly |
21
F igure 1. C alibration of Sucrose G radients
Two 34 m l 15-60% su cro se gradients w ere prepared and
co llected as describ ed in Chapter II. Each fraction was
m easu red for its refractive index on a refractom eter.
FRACTION
REFRACTIVE INDEX
W
u
K >
K > ,
■o
%09 - r ° ° r r ’'
in serted into th is holder, the needle was raised until it had
cleanly p ierced the bottom of the tube. The gradient was then
pumped out through this hollow needle via a Buchler p olystaltic
pump at a rate of 2 m l per m inute, flowing through an Isco UV
A nalyzer before being co llected on an Isco fraction co llecto r
(Isco, L incoln, N ebraska). The absorbance was m onitored at
254 nm and recorded on an 8 inch chart record er. All gradients
w ere co llected in 1 m l fra ctio n s. J
I
When polysom e hom ogenates w ere fractionated on th ese |
i i
gradients, fraction s of heavy polysom es and m edium p olysom es
i j
w ere pooled as indicated by the gradient p rofile (see Table 2).
I !
These polysom e fraction s w ere then co llected and prepared for j
storage in the sa m e m anner as whole p o ly so m es. 1
i i
l
| Incubation of T issu e S lices
i ;
j ,
| S lices of skin, or p ie c e s of wings and le g s from chick em bryos ]
I i
j
w ere incubated at 0. 5 g p er 5 m l of E agle's Minimum Medium
(obtained from G ibco, Grand Island, New York) supplem ented with I
100 u g /m l sodium ascorb ate (82). After incubation at 37° C for
I
i
; 30 m in u tes, the m edium w as rem oved by aspiration and rep laced
! i
i
^ j
with 5 m l of fr e sh m edium plus 50 uCi of H prolin e. j
Incubation was continued fo r an additional 30 m inutes; the m edium
was again rem oved by asp iration and the tissu e s w ere hom ogenized
in 2 m l of H om ogenizing Buffer, after which the supernates w ere
analyzed on su crose gradients.
Incubation System for P rotein Synthesis
In stu d ies designed to determ ine the optim um conditions
for p rotein sy n th esis, 1-2 A2 6 0 units of p olysom es w ere
incubated at 37°C for 30 m inutes in a total volum e of 100 ul
I
I
containing: T ris-H C l, 20 mM , pH 7. 4 at 37° C; A TP, ImmM;
I
G TP, 0 . 2 mM; creatin e phosphate, 15 mM; creatin e j
j
j phosphokinase, 0. 5 units; 19 am ino acids (excluding prolin e), |
0. 1 mM; ^H proline, 5 uCi (sp ecific activity i 30 C i/m m ole).
; 1
U n less stated oth erw ise, the incubation m ixture a lso contained: '
j !
! X • 1 I
K , 90 m M (skin) or 150 mM (wing and 1 eg) ; Mg T , 4. 5 mM |
(skin) or 7. 5 mM (wing and leg) ; Initiation F a cto rs, 1 m g /m l; i
' " s
I ASyQ enzym es; 2m g/m l; and DTT, 1 mM. I
j I
A 10 X amino acid solution was prepared in T ris-H C l buffer j
|
| and sto red frozen. An energy generating sy ste m containing ATP,
j
i G TP, C P, and CPU was prepared in the sam e buffer at a tenfold
|
concentration and stored frozen .
!
The incubation sy stem was alw ays made up in the sam e order:
, I
H p ro lin e, amino a cid s, buffer, energy sy ste m , ASyo, IF, and 1
; |
after a 10 minute pre-incubation, p oly so m es. Incubation was j
tim ed fro m the addition of p o ly so m es. I
A ssa y for the Incorporation of P rolin e
P rotein syn th esis in the incubation sy stem was stopped by the
addition of 2 volum es of 1 M T ris buffer (pH 10) and further
incubation at 37° C for 10 m in u tes. T his rem oved amino acids
from charged tRNA m o le c u le s, and thereby prevented
unincorporated lab el from being included in the sam p les. The
sam ples w ere then tran sferred into 2 m l of ic e cold w ater,
| follow ed by the addition of 50 or 100 ug BSA and 2 m l of ic e cold
10% TCA (T rich loroacetic acid ). The BSA served a s a ca rrier to
fa cilitate precipitation. P recip ita tes w ere co llected on g la ss
fiber filters (Whatman G F /C ) which w ere w ashed 3X with 5% TCA
1 and 3X with cold 95% ethanol. The filte r s w ere dried, placed in
scin tillation v ia ls , and covered with 0. 5 m l of NCS tissu e
solu b ilizer (A m ersh a m /S ea rle, A rlington H eights, 111.) to
d isso lv e the precip itated p rotein in order to obtain better
j counting efficien cies (101, 102). To each v ia l was added 3 m l of
| a scin tillation cocktail co n sistin g of 0. 38 g POPOP and 15.2 g PPO
| per gallon of toluene. Since th ere is a sm a ll in crea se in
| counting efficien cy with a g rea ter volum e of cock tail, studies on
the translation rates of p o ly so m es w ere counted in 10 m l of
scin tillation cocktail.
In a ssa y s on the r e le a se of new ly syn th esized protein s,
> the above procedure was slig h tly m odified. The pH 10 m ixture
was put into 2 m l of ic e cold w ater and then filtered through a
a n itro cellu lo se filter (M illipore C o ., Bedford, M a ss.) which
retained the p olysom es and th eir nascent protein chains; the
relea sed soluble proteins w ere in the filtra te which was
precipitated as above and co llected on glass fiber filte r s.
In studies using heavy p o ly so m es, the product was expected
to be la rg ely collagen, and th erefo re the 10% TCA solution was
augm ented with 0. 5% tannic acid to ensure the com plete
p recip itation of collagen.
A ssay for T ranslation Tim e
The incubation sy stem w as in crea sed to 500 ul with all
com ponents at the sam e concentrations as d escrib ed before,
u n less stated oth erw ise for a particular experim ent. To
m easu re the tran slation tim e (the tim e for the ribosom e to
tra v erse the mRNA from the 5' end to the 3* end and rele a se
the protein chain) r e-in itia tio n of rib o so m es on the 5' end of
the polysom e mRNA was blocked by the addition of ATA
(aurintricarboxylic acid) at a final concentration of 0.1 mM
<103, 104).
Incubation began with the addition of p o ly so m es, and sam ple
aliquots of 20 or 50 ul w ere rem oved from the incubation sy stem
at 1 or 2 m inute in tervals and a ssa y ed for the incorporation of
proline into r elea sed soluble protein.
27
C ollagenaae A ssa y of N ewly Synthesized P rotein s
A 500 ul incubation m ixture was divided after a 30 m inute
incubation into two equal vo lu m es. To one was added 50 ul of
buffer, and to the other 50 ul of C lostridium histolyticum
co llagen ase prepared by the m ethod of Benya et a l. (105, 106).
The collagen ase solution had a concentration of 2 m g /m l.
Incubation was continued for an additional 30 m in u tes, and the
soluble protein was then assayed for the incorporation of radio
activ ity as described above. The d ifference in CPM between the
control sam ple and the collagen ase treated sam ple was taken as a
m ea su re of the amount of collagen syn th esized (37, 78, 79).
C ollagenase A ssay of P o ly so m e F ractions
A volum e of 200 ul of heavy polysom e solution was incubated
with either 50 ul of buffer or 50 ul of co llagen ase. Incubations
w ere for 30 m inutes at 4° or 37° C, after which the m ixture was
added to 1. 5 m l cold buffer and la yered on top of a su cro se
gradient for further a n a ly sis.
P olyacrylam id e Gel E lectrop h oresis
P olyacrylam id e g els w ere prepared after the method of
Sakai and G ross (107), a s m odified by L apiere et al. (49). The
running g els w ere 7. 5%, and the stacking g els w ere om itted.
E lectro p h o resis was perform ed with a constant current of 3 m A
per g el for 2 1/2 to 3 h ou rs. A ll the reagents for acrylam ide
gels w ere electrop h oretically pure (obtained from B io-R ad
L ab oratories, Richmond, C alifornia). G els w ere stained o v e r
night in a 12. 5% TCA solution containing 0. 25% C oom assie
B rillian t Blue R -250. G els w ere destained overnight in 12. 5%
TCA and stored in 7% a cetic acid .
A nalysis of collagen sam ples from the incubation sy stem was
perform ed as fo llo w s. After treatm ent with pH 10 buffer the
en tire m ixture was added to 4 m l of a pH 4. 8 acetate buffer
(20 mM sodium acetate, 1 M urea) containing 0. 4 mg of
j
: denatured ca rrier collagen. The m ixture was then centrifuged
|
on the SW 39 rotor in a Beckm an Model L centrifuge at 25,000
|
I
rpm for 150 m inutes. The supernate was dialyzed overnight
; against the sam e buffer, and the dialysate w as then adjusted to a
i
: pH of 3. 5 with a cetic acid. The solution was then m ade 5% in
i
; sodium chloride to precip itate the collagen. The p recip itate was
i co llected by centrifugation at 15, 000 x g for 20 m in utes,
resuspended in the sam e buffer and denatured at 60°C for
30 m in utes. Su crose was added to a fin al concentration of 25%.
The sam ples w ere la yered on the gels in volum es of 50 ul or 100 ul.
Follow ing electro p h o resis the g els w ere stained, destained,
and slic e d into 3 m m sectio n s. Each sectio n was incubated in
1 m l of NCS tissu e so lu b ilizer at 60°C for two hours. This
caused the g el s lic e s to sw ell to about 4 tim es th eir original
volum e, perm itting the proteins to diffuse out (101). The NCS
treated gel sam ples w ere tran sferred into scin tilla tio n via ls and
counted in 10 m l of a scin tillation cock tail. Counting was done on
a Beckm an L S-233 scin tillation counter.
D eterm ination of Sedim entation V alues
The calculation of sedim entation valu es for su cro se gradients
| has been sim plified by the publication of a s e r ie s o f tables for I,
I
! the integration constant in the follow ing ex p ressio n for the
' sedim entation constant:
^ O , w t J 2 (t2 - t i) = I (z2) - I (Z l)
w here z is the su cro se concentration at any part of the gradient. |
i
T ables for I have been computed to cover a va riety of tem p eratu res, j
i
d en sities, etc. (108). i
i '
Table 1 lis t s the sedim entation valu es a s calculated by this J
| m ethod for p a rticles that have been sedim ented on a 34 m l 15-60% j
su cro se gradient at 27 ,0 0 0 rpm for 90 m inutes on an SW 27 rotor.
Although the tab les have been v erified by the author against a wide '
1 i
I
i v a riety of p a rticles with known sedim entation constants, th eir u se
in th ese studies is intended only to provide a 'best estim a te1 . |
The only internal calibration m arker for the values listed in Table 1 ;
is the 80 S m onosom e peak which w as e a sily distinguished in
F raction 30. 3 of m o st gradients. It is rea ssu rin g that the
30
Table 1
S Values for F raction s of 15-60 % Sucrose Gradient
action % Sucrose S
1 5 9.34 5, 000
2 57.98 4, 000
3 56.62 3,320
4 55.26 2,835
5 53.90 2,4 2 0
6 52. 54 2,0 9 0
7 51. 18 1,800
8 49. 82 1,580
9 48.46 1,390
10 47. 10 1,225
11 45. 74 1.090
12 4 4.3 8 970
13 4 3.0 2 870
14 41.66 775
15 40. 30 695
16 3 8.9 4 625
17 37. 58 560
18 3 6.22 505
19 34. 86 450
20 33. 50 405
21 32. 14 360
22 30. 78 320
23 29. 42 290
24 28.06 250
25 26. 70 220
26 25. 34 190
27 23. 98 164
28 22.6 2 137
29 21.2 6 113
30 19. 90 90
31 18.54 67
32 17. 18 46
33 15.82 28
34 14. 46 6
31
p redicted value for th is F raction according to Table 1 is also
j 80 S.
I
!
j E stim ation of the Number of R ibosom es per P olysom e
j
It has been shown in a number of studies that a polysom e {
I
j
c a rr ie s one ribosom e per 90 nucleotides of mRNA (76, 109, 110). j
Hans N oll has derived a linear relationship betw een the
| sedim entation constant of a polysom e and the num ber o f rib osom es
! on it, which can be ex p ressed as
I I
lo g Sn = log Si + 0 .6 0 log n j
w here Sq is the sedim entation constant of a polysom e with n
rib osom es (76). This relationship has b een verified by N oll for
polysom es with up to 20 rib osom es.
To determ ine the number of ribosom es for a polysom e at any
> part of the gradient, the S value for that fraction was obtained j
j !
from Table 1, and the number of rib osom es calculated from : j
I
j n = antilog (log - log S i) /0 . 60.
j One can a lso m ake an estim ate of the number of rib osom es for any
| given mRNA if the s iz e of the protein tran slated from it is known.
As an exam ple, m yoglobin contains 153 am ino a c id s, and the mRNA <
for m yoglobin m ust contain at le a st 459 nu cleotid es. Dividing
| j
this la st value yield s a value of 5 rib osom es per polysom e, which i
has b een confirm ed experim entally (99).
: j
l . . 32
D eterm ination of the T ranslation Tim e
The translation tim e of an mRNA is the total tim e required
for a ribosom e to attach to the initiator site at the 5' end of the
m essen g er RNA, m ove down the mRNA to the 3' end, and
d isso cia te from the m essen g er w hile relea sin g the newly-
synth esized protein chain. T ran slation tim e should be
distinguished from 'tran sit tim e 1, the tim e it takes a ribosom e to
j tr a v e rse the mRNA, and thus excluding the tim e required for
|
| initiation and relea se. Since the tim es required for initiation
j and relea se a re quite short (99), the differen ce betw een tra n s-
! lation tim e and transit tim e b ecom es in sign ifican t for very large
! j
p olysom es. |
A s d escrib ed in a previous section , tran slation tim e w as j
I
m easu red as the appearance of r elea sed protein under conditions
i |
I w here re-in itiation w as blocked. The v a lu es determ ined by this !
| i
I procedure a r e then interm ediate betw een tran slation tim e and |
: i
tra n sit tim e. Since the oollagen p olysom e is exceptionally la rg e, j
!
th ese experim ental valu es can b e used a s a good approxim ation for j
! J
eith er the translation tim e or the tran sit tim e.
To determ ine the tran slation tim e fo r collagen p o ly so m es,
th ey w ere incubated w ith an inhibitor o f re-in itia tio n . Thus new
rib osom es oould not attach at th e 5' end (111), but a ll the
rib osom es alread y on the m e sse n g e r could m ove fr e e ly to the 3* end
and r e le a se their com pleted protein chains into the solution (103).
By assayin g for the incorporation of lab el into the relea sed
I
i p rotein s, the tim e at which the la st ribosom e is r elea sed from
i
| the polysom e is the tim e at which no m ore lab elled protein appears
| in solution. This is the tim e, th erefore, when the amount of
1
lab elled protein reach es a plateau le v el.
CHAPTER HI
RESULTS AND DISCUSSION
Size of C ollagen P olysom es
A p relim in ary study was done to exam ine the spectrum of
polysom es in chick em bryo connective tis s u e s . Sam ples of
skin, and of wings and leg s w ere incubated in E agle's M inimum
Medium with tritiated proline for 30 m inutes at 37°C. F igure 2
shows the sedim entation p ro files of th ese p olysom es and the
radioactivity incorporated into nascent protein chains on the
p o lysom es. It can be seen that polysom es from both sam p les
had a wide range of s iz e s , up to 2000 S. The skin polysom e
p rofile showed a v ery pronounced peak of heavy p olysom es with
an average sedim entation constant of about 1400 S. The peak of
proline incorporation was a sso cia ted with polysom es of about
1600 S. The wing and leg polysom es incorporated proline to
a le s s e r degree and this m ay have been due to the fact that th ese
tissu e sam ples w ere too large to perm it rapid inward diffusion of
the radioactive proline during the incubation. Since only a part
of th e heavy polysom e fraction from skin showed a high rate of
F igure 2. Sedim entation P ro files of L abelled P olysom es
From Chick Embryo Connective T issu es. T issu e s were
! incubated in vitro in E agle's Minimum Medium with 50 uCi
3
H proline for 30 m inutes. P olysom es w ere sedim ented on a
j 30 m l 15-60% su cro se gradient. P oly so m es at the bottom of the
i
gradient (Fraction 1) have a sedim entation constant greater than
i
2000 S. Fig. 2A: polysom es from wings and le g s . Fig. 2B:
p olysom es from skin; the peak of the heavy polysom e fraction
is about 1400 S, and the peak of incorporated p rolin e is on
p olysom es of about 1600 S.
(--------) ABSORBANCE (254 nm)
M
M
M
M
M '
tu,
M
N
M
(--------- ) 3 h p r q u n E INCORPORATED CPM 10"2
proline incorporation, it is probable that m o st of the polysom es in
this broad peak w ere not involved in collagen syn th esis.
To im prove the resolvin g power of the grad ien ts, their volum e
was in creased to 34 m l, and subsequent tis s u e sam ples were fir s t
fractionated into non-detergent and d etergen t-treated hom ogenates
before th ey w ere applied to the gradient for an a ly sis. The
resu lts o f these m odification s are illu stra ted in F igu res 3-5.
Figure 3 shows the sedim entation profile of a skin sam ple
hom ogenized without detergent. A sharp m onosom e peak
(F raction 30) and two m edium polysom e peaks (Fractions 24 and
18) w ere evident. No heavy polysom e peak was p resen t, but th is
m ight have been expected sin ce collagen p olysom es a re m em brane-
bound (37). In F igure 4 a sim ila r p rofile is shown for a sam ple
hom ogenized in the p resen ce of deoxycholate and Triton X-100.
The u se of th ese detergents for releasin g polysom es from the
m icro so m a l m em branes has been dem onstrated (90, 112-115).
This procedure has been reported to in c r ea se the y ield of RNA
extracted to about 60-67% of the total cellu la r RNA (90, 116).
C om parison with F igure 3 shows that the m onosom e peak and
the two m edium p o l y s o m e peaks w ere p resen t in the sam e
fractions of the gradient. In addition th ere is a distinct peak of
heavy p olysom es (F raction 8) made soluble by the d etergen ts.
F igure 3. Sedim entation P ro file of P olysom es from a non
detergent hom ogenate of chick skin. A 4 m l sam ple was
la y ered on a 34 m l 15-60% gradient. The m onosom e peak
appears in F raction 30, and two m edium sized polysom e peaks
are centered around F raction s 24 and 18. No heavy polysom e
peak can be seen .
39
ABSORBANCE (254 nm )
2.0
1.5
1.0
0.5
FRACTION
F igure 4. Sedim entation P ro file of P oly so m es from a
detergent hom ogenate of chick skin. A 2 m l sam ple was
layered on a 34 m l 15-60% gradient. The m onosom e
peak, and the two m edium peaks appear as in F ig. 3. In
addition th ere is a definite peak of heavy polysom es
centered about F raction 9.
FRACTION
ABSORBANCE (2 5 4 nm )
C M CM CM
CM
F igure 5. Sedim entation P ro file of P olysom es from a
detergent hom ogenate of chick skin. A 4 m l sam ple was layered
on a 34 m l 15-60% gradient. Only the bottom two thirds of the
gradient are shown h ere, sin ce the absorbance at the top was
off sc a le . With the la rg er sam ple volum e, the polysom e peaks
are displaced sligh tly towards the bottom of the gradient. The
!
I heavy polysom e peak is s till c lea r ly d iscern ib le.
43
FRACTION
ABSOR BANCE( 2 54 n m T
U W
u
*o
o -
F igu re 5 shows that the heavy polysom e fraction could still be
reso lv ed , even when the sam ple volum e layered on the gradient was
in crea sed to 4 m l.
Table 2 su m m arizes data from 50 different su c ro se gradients,
using sam ples from different hom ogenates and of different volu m es.
Except when v ery la rg e sam ple volum es (4-6 m l) w ere used , the
sedim entation p ro files w ere v ery reproducible.
Both Manner et a l. (72) and Fernandez-M adrid (73) reported
evidence of polysom e fractions a s large as those shown in
F igu res 2 and 3. H ow ever, n eith er was able to ach ieve v ery good
resolu tion of this fraction due to technical p rob lem s. L azarid es
and Lukens (77) a lso u sed chick em bryo wings and le g s , and
reported that th eir la r g est polysom e fraction was only 300-400 S.
It can be clea rly seen in Figure 2 that much la rg er p olysom es
w ere obtained from the sam e tis s u e s . The reason for the
d ifferen ce in th ese resu lts is that L azarides and Lukens
sedim ented th eir p olysom es on a 15-40% su cro se gradient at
41, 000 rpm , w hile in th is study polysom es w ere sedim ented on a
15-60% gradient at 27, 000 rpm. Thus the heavy polysom e
fraction that w as near the bottom of the gradient in F igure 2
could not have been detected by L azarides and Lukens sin ce it
would have been p elleted on the bottom of the centrifuge tube with
th eir procedure.
________ _______________ 45
Table 2. P olysom e D istribution on Sucrose G radients.
T his data rep resen ts 50 different gradient p r o files. A ll gradients
w ere 34 m l 15-60% su cro se gradients and prepared and collected
as d escrib ed in Chapter H. The fir s t number in each colum n
rep resen ts the fraction (s) in which that particular cla ss of
p o ly so m es was found, and the value in the p arenth eses rep resen ts
the number of gradients used to m ake that determ ination. Each
sam p le number id en tifies the volum e and nature of the sam ple:
2 D m eans 2 m l of detergent hom ogenate; 4 ND m eans 4 m l of
non -detergen t hom ogenate. OS ind icates that the absorbance was
off sc a le for that region of the gradient. See F igu res 3-5 for
exam ple of sedim entation p ro files.
Table 2
P olysom e D istribution on Sucrose Gradients
•
Heavy Heavy Peaks Middle M iddle Peaks, M onosome
Sample # Region I n Region I H Peak
1 D 4 1-11 (3)
----- —
12-27 19. 5 (2) 27 (1) 30 (3)
2 D 1 1-12 (1)
-----
8 (1) 13-26 18 (1) 24 (1) 30 (1)
3 D 12 1 -1 0 .5 (12) 4 (2) 7. 8 (8) 12-26 18.1 (8) 2 3 .9 (7) 29 (3)
4 D 11 1 -1 0 .2 (11) 4 (1) 7. 5 (8) 11-26 19 (6) 26 a) OS
5 D 2 1-10 (2)
-----
7 (2) 11-26 20 (2) OS OS
6 D 2 1-8 (2) 4 (1) 8 (1) 9-26 OS OS OS
1 ND 3 1-12 (3)
-----
8 (1) 13-26 21 (2) 2 6 .5 (2) 31 (2)
2 ND 2 1 -1 2 .5 (2)
-----
-----
13-26
----- -----
OS
3 ND 10 1-12 (10)
----- -----
13-26 19 (4) 25. 1 (10) 31 (7)
4 ND 3 1 -1 1 .3 (3)
----- -----
12-26 18 (3) 24 (1) OS
50 1 -1 0 .9 (49) 4 (4) 7 .6 (21) 12-26 18.9(28) 2 4 .9 (23) 30.3 (16)
*
Table 3
D istribution of R ibosom es on D ifferent P o ly so m es
Number of R ibosom es
P olysom e F raction
Heavy
Medium I
Medium
Peak Ranee
116 78-170
15-16 10-53
5 - 6 4-8
n
Values are determ ined from the gradient p rofile of F ig . 4,
and calculated a s d escrib ed in M ethods.
48
The sedim entation constant of any polysom e fraction can
be reasonably estim ated by its position in the gradient. Such
valu es w ere calculated and are shown in Table 1. Knowing
the value of the sedim entation constant, the number of
rib osom es on any given polysom e can a lso be determ ined.
The number of rib osom es was calculated for each of the three
polysom e fraction s of Figure 4, and are liste d in Table 3.
The sm a ller of the m edium p olysom es contained about 5-6
rib o so m es, and probably contained mRNA for hem oglobin sin ce
that is the size reported for globin p olysom es (99). P olysom es
in the second m edium fraction contained from 10 to 53 rib o so m es.
Such a range could include m any different sp ecies of mRNA,
including p olysom es coding for a single collagen p ro- CL chain
which would be expected to have about 42 rib o so m es. The
heavy polysom e peaks contained about 116 rib o so m es, and th is is
approxim ately the number of rib osom es predicted for a
p o ly cistron ic m e ssen g er RNA coding for a ll 3 pro - CL chains
that constitute the trip le stranded p rocollagen m o lecu le.
E xcept for the m onosom e peak, the polysom e fractions
rep resen ted in F igu res 2 -4 appeared in broad bands. This
spreading of a polysom e fraction on the gradient is caused by
two fa c to r s. A com bination o f the original sam ple volum e and a
sm a ll amount of diffusion help to spread a polysom e band. It
has a lso been shown that polysom es are not uniform ly saturated
with rib o so m es. P alm iter (99) exam ined ovalbum in p oly so m es,
which have a capacity for 13 rib o so m es. Under norm al
conditions the mRNAs for ovalbum in m ay carry from 7-12
rib o so m es, and ovalbum in p olysom es consequently sedim ent in a
broader band on a gradient. Thus the heavy polysom e fraction
has been interpreted as co n sistin g, in part, of polysom es
containing the sam e collagen mRNA throughout its sedim entation
range.
I Studies on P rotein Synthesis in the C e ll-F r e e System
i
Conditions for the c e ll-fr e e sy stem capable of collagen
j
i
| syn th esis have been described for polysom es from chick em bryo
I
wings and leg s (77), but not for p olysom es from chick em bryo
skin. It was th erefore n e c e ssa r y to perform a s e r ie s of
experim ents testin g the in vitro sy stem for optim um activity
| in relation to different ionic concentrations that affect the
I
I
j stab ility of rib o so m es, and the various protein factors needed to
stim u late protein syn th esis under th ese conditions. The sam e
i studies w ere perform ed with the wing and leg polysom es and th ese
I
served as a standard for com parison.
In F igu res 6-15 are presented experim ents in which a ll
j
I com ponents of the c e ll-fr e e incubation system w ere optim um
i_ _ _ _ _ _ _ _ . . . . . . . . . . . . . . . . . . . . . . . . . . 50
except for the sin gle factor being tested . F igu res 6 and 7 show
the effect of Mg** on protein syn th esis with skin p olysom es, and
with wing and leg p o ly so m es. Both sy stem s had d istin ct
optim um requirem ents for M g'*"*'. Skin polysom es w ere m ore
sen sitiv e to the Mg concentration, and had a significantly
low er optim um . The optim um Mg++ concentration for wing and
leg polysom es of 7. 5 mM was iden tical to the value reported by
L azarides and Lukens (77), while the optim um of 4. 5 mM for
| skin was in agreem ent with studies on many other system s
| (104, 113, 117, 118).
The effect of K* is shown in F igu res 8 and 9. Although an
optim um concentration could be d iscern ed for both system s
| neither skin polysom es nor wing and leg p olysom es w ere as
I . + 4-
sen sitiv e to K+ as they w ere to Mg . H owever, the ratio of
optim um K+ to optim um Mg++ was the sam e for both system s
with a value of 20:1. This ratio of ion ic concentrations has been
| reported as being id ea l for the stab ility of ribosom e s (119).
A group of essen tia l fa cto rs required for protein synth esis
is contained in the c e ll sap. Included among th ese are tRNAs
I
! and tRNA-am ino a cy l sy n th etases, at le a st two elongation
fa cto rs, and p o ssib ly a polypeptide relea sin g factor and a ribosom e
d isso cia tio n factor (94, 114, 120, 121). E a rlier studies used the
| c e ll sap d irectly , but such preparations often contained RNAses j
I __________ . ____ ____________ _ . . . ................... _ _51
F igure 6. The E ffect of Mg++ on P rotein Synthesis with
P olysom es from Chick Skin. The Mg+* concentration is
fa ir ly critica l, and protein syn th esis d eclin es rapidly on either
sid e of the optim um range.
’h proline incorporated
CPM-10"3
© o
O t
o
o > ^ 0 0
b o b
■ » 1
o
o
b
z
•D
♦
♦
3
z
»•
yi
w
F igure 7. The E ffect of Mg++ on P rotein Synthesis with
P oly so m es from Chick Wings and L egs. This system is le s s
sen sitiv e to Mg++ concentrations than the one derived from
skin (see Fig. 6).
3H PROLINE INCORPORATED C P M 1 0 '3
M w
m
U 1
vn
F igure 8. The E ffect of K+ on P rotein Synthesis with
P olysom es from Chick Skin. Although th ere is an optimum
protein syn th esis with K+ around 90 m M , protein syn th esis can
be m aintained for a wide range of potassium concentrations.
H PROLINE INCORPORATED
CPM 10“ 3
U l O l
F igure 9. The E ffect of K+ on P rotein Synthesis with
P olysom es from Chick Wings and L egs. The optim um
concentration is at 150 mM, though protein syn th esis can be
m aintained over a wide range of K '* ' concentrations.
mM
3h p r o l i n e i n c o r p o r a t e d C P M 1 0 “ 3
O t
o
n
♦
N
or other inhibitors and th ese factors are now standardly purified
by precipitating them at a pH of 5. 2, or with an am m onium sulfate
fractionation betw een 40% and 70% of saturation. There is still
considerab le controversy about the efficacy of th ese two m ethods.
H om ologoue preparations of pH 5 enzym es w ere tested for
their ability to stim ulate protein syn th esis in the two sy ste m s.
F igure 10 show s that the pH 5 enzym es prepared from skin failed
to stim ulate protein syn th esis on skin p o ly so m es, and in fact
exhibited a m arked inhibition at higher concentrations. F igure 1 1
i
i illu stra tes that pH 5 enzym es prepared from wings and leg s
j
doubled the rate of protein syn th esis on wing and leg p olysom es
at an enzym e concentration of 4 m g /m l. Even at half of this
concentration the pH 5 enzym es had a significant stim u latory
effect. The d ifference in the effect of pH 5 enzym es on the two
| sy stem s is both significant and rem arkable. It is not lik ely that
| this difference can be attributed to a procedural failu re in the
I
preparation o f the skin pH 5 solution, sin ce both enzym e fractions
w ere prepared on the sam e day, using the sam e solu tion s, and
from tissu e s obtained from the sam e em bryos.
Hom ologous preparations of A S j q enzym es w ere tested under
sim ila r conditions. It can be se e n in F igu res 12 and 13 that the
rate of p rotein syn th esis w as alm ost trip led at the optim um
concentrations in both sy ste m s. At the high est concentrations
tested , the ASyp enzym es from wings and le g s w ere le s s than
optim um , and th is m ay reflect an inhibitory component p resen t
in this fraction. Since the AS7 0 enzym es at optim um
concentrations stim ulated protein syn th esis to a greater degree
in both sy ste m s, they w ere u sed in a ll other studies of protein
syn th esis in vitro .
In addition to the soluble factors from the c e ll sap that
fa cilita te the m ovem ent of rib osom es along the mRNA and the
elongation of the nascent polypeptide chain, another group of
protein factors are required to prom ote the attachm ent of
rib osom es to the mRNA in form ing an initiation com plex. At
le a st th ree d istin ct initiation factors have been identified in
preparations derived from 'w ashed' rib osom es (98, 114, 117,
118, 120, 122-124), and one study reported 4 initiation factors (125).
B ecause th ese initiation fa cto rs are found a sso cia ted with
rib o so m es, they w ere iso la ted by washing rib osom es in a 0. 5
M KC1 solution, and concentrated from the supernate follow ing
high speed centrifugation to p ellet the rib o so m es. It would be
reasonable to expect that a portion of th ese initiation factors are
in solution in the c e ll sap, and one report claim ed that they can
be found in the pH 5 enzym e fraction (121).
61
A low rate of syn th esis can occu r in the ab sence of
initiation factors as th ose rib osom es alread y attached to the
p olysom al mRNA m ove towards the 3' end, but re-in itia tio n is
n e c essa ry for continued protein sy n th esis. F igu res 14 and 15
show that the rate of protein sy n th esis was in crea sed dram atically
in both sy stem s with optim um concentrations of initiation fa cto rs.
The rate in crea se was sevenfold for the wing and leg p o ly so m es,
and fourfold with skin p olysom es w here an in crea se in both the
nascent and r elea sed proteins indicated that re-in itia tio n on the
p olysom es occu rred and that com plete protein chains w ere
| relea sed . At the highest concentration tested , the initiation
I
■ factors from skin becam e le s s effectiv e, indicating that som e
!
inhibitory factor m ay have been p resen t in this fraction.
Table 4 su m m arizes the data for the studies rep resen ted
in F igu res 6-15. The two incubation sy stem s had considerably
different requirem ents for optim um protein sy n th esis. The
m ost striking and in terestin g d ifferen ces w ere found with the
different preparations of enzym es and fa cto rs. It is apparent
that there was in each sy stem at le a st one inhibitory factor
j contained in the preparations of soluble enzym es or initiation
! fa cto rs. The literatu re contains m any referen ces to such an
inhibitory factor(s) which is u su ally found in the ribosom al salt
wash preparation of in itiation fa cto rs (114, 123) or in the cell
sap from which soluble factors are prepared (99).
The d ifferen ces betw een the inhibitory effects seen in the tw o
sy stem s can be explained by proposing that each tissu e contains
an inhibitory factor with different ionic character such that they
would not precip itate or 'sa lt out' under the sam e conditions.
This was apparently the ca se sin ce the inhibitor in the skin sy stem
was precip itated as part of the pH 5 fraction, but was not
! precipitated as part of the A S j q fraction . E xactly the rev e r se
\ was seen with the wing and leg sy stem . It is a lso quite p o ssib le
i
| that the inhibitor found in the pH 5 fraction of skin was the sam e j
as that found in the initiation factor preparation.
Since inhibitors in the different tissu e s have been shown to have j
• different precip itation p ro p erties, this m ay account for p referen ces j
i •
! in the m ethod of preparation of the cell sap factors in different
I
la b o ra to ries. It a lso shows the need to evaluate the effect of
different preparations on a polysom e sy stem so that th eir effects
do not m ask the activity of the polysom e preparation its e lf. For
th ese reason s enzym e preparations from the sam e tissu e serv e as
a starting point for standardizing a protein syn th esis sy stem before
| heterologous preparations can be m eaningfully em ployed. This
I la s t point has been illu strated by Heywood and colleagu es who have
shown that both m yosin mRNA (87) and m yoglobin mRNA (126)
F igure 10. The Effect of pH 5 E nzym es on P rotein Synthesis
with P olysom es from Chick Skin. This enzym e fraction not only
fa ils to stim ulate protein syn th esis, but clea rly causes it to be
d ep ressed .
64
3 h proline incorporated
CPM-10‘3
^ M U «
T J
X
U l
m
z
IM
-<
5
m
(/>
3
•Q
-v.
3
F igu re 11. The E ffect of pH 5 E nzym es on P rotein
Synthesis with P oly so m es from Chick W ings and L egs. The
addition of th is enzym e fraction clea rly in crea ses the le v e l of
protein sy n th esis.
p H 5 ENZYMES Cmg/ml)
!
3H PROLINE INCORPORATED CPM 10"3
O '
F igure 12. The E ffect of ASyg Enzym es on P rotein Synthesis
with P olysom es from Chick Skin. Although the optim um
stim ulation is achieved at an enzym e concentration of 5 m g /m l.
the addition of enzym es at a concentration of 2 mg /m l is clea rly
sufficient.
68
AS7 n ENZYMES (mg/ml)
3H PROLINE INCORPORATED CPM 10"3
o
u
b
b
w
b
O '
v O
F igure 13. The Effect of ASyg E nzym es on Protein Synthesis
with P oly so m es from Chick Wings and L egs. Optimum
stim ulation is achieved with enzym e concentrations of 2 -4 m g /m l.
An inhibitory effect begins to m an ifest its e lf at enzym e
concentrations of 5 m g /m l or higher.
70
ENZYMES (m g /m l)
3H PROLINE INCORPORATED C P M 10’ 3
U t
N |
K >
>
c />
N J
o
m
F igure 14. The E£fect of Initiation F actors on Protein
Synthesis with P olysom es from Chick Wings and L egs. The
optim um syn th esis occu rs with IF concentrations of 4 m g /m l,
but a concentration of 2 m g /m l was considered sufficient for
m ost stu d ies.
n
i
o
2
a.
o
o
U J
I-
<
oe
O
a.
o c
O
o
z
o
o c
a.
x
(* »
4-
3-
4.0 3.0 X0 to
INITIATION FACTORS
(mg/ml)
73
F igure 15. The E£fect of Initiation F actors on Protein
Synthesis with P olysom es from Chick Skin. The le v e l of
incorporation of p rolin e was m easu red for relea sed proteins
and for nascent protein chains. Optimum syn th esis occu rs at
IF concentrations of 1 to 1. 25 m g /m l, and an inhibitory effect
occu rs at higher concentrations.
NASCENT
R E H A S H
30
CM
O
25-
20 -
U J
15-
10 -
1.5! 1.25 0.75 M >
INITIATION FACTORS (mg/ml)
0.25
75
Table 4
Dependancy of the C e ll-F r e e System on Various F actors
F actors
Incubation System
Skin Wing & Leg
K+ : Optimum
Range
90 mM
40 - 160 mM
150 mM
120 - 200 mM
Mg++ : Optimum
Range
4. 5 mM
2 .4 - 6. 5 mM
7. 5 mM
4. 5 - 9. 5 mM ,
pH 5 : Optimum
Range
Inhibition
None
None
Y es > 0.1 m g /m l
4 m g /m l
1.8 - 4 . 0 m g /m l
No
AS?0 :
Optimum
Range
Inhibition
5 m g /m l
1.5-5 m g /m l
No
i
2 m g /m l
2 - 4 m g/m l j
Y es > 4 m g /m l
I F : Optimum
Range
Inhibition
1 m g /m l
1 - 1 .2 5 m g /m l
Y es i 1 .5 m g /m l
4 m g /m l
1 . 5 - 4 m g /m l
No
l
i
I
The Range is defined as that range of concentrations where
protein syn th esis is at le a st 80% of the optim um le v e l.
Inhibition sig n ifies that an in crea se in concentration of an
enzym e resu lts in a significant d ecrea se in protein
sy n th esis.
req uire tis su e -sp e c ific initiation factors for continued protein
sy n th esis.
The data in Table 4 a lso show that the two hom ologous sy stem s
had equivalent ranges for the optim um concentrations of the
different factors tested . When a ll conditions w ere optim ised,
the two sy stem s w ere equally efficient in protein sy n th esis.
Studies on C ollagen Synthesis with Isolated P olysom e Fractions
The two m ajor polysom e fractions from skin w ere sep arated on
j gradients and collected as described in Chapter II. To detect
i
| the p o ssib le p resen ce of contam inating m a teria l in th ese polysom e
I fra ctio n s, they w ere analyzed by absorbance spectroscop y. I
F igure 16 shows the u ltraviolet absorbance spectrum for the '
' !
j two fraction s. The absorbance spectra indicated that both |
preparations w ere reasonably fr e e of contam ination. A standard
criterio n for ribosom al purity is the -^ 260^ 280 rati°* with a
value of about 1. 85 being m ost com m on for pure rib osom es (127).
H ow ever, p olysom es contain som e nascent proteins and at le a st
two additional protein m o ieties whose function is still ob scu re
(128,129). Thus the absorbance ratio could be expected to be
j som ewhat below 1. 80 for p olysom es. Both polysom e fractions
I
shown in F igure 16 had an A2^q/-^280 °*
j
Since both the heavy polysom e fraction and the m edium
polysom e fraction contained polysom es su fficien tly large
i_ _ _ _ _ _ _ _ _ _ _ 77
to sy n th esize collagen pro- CL chains, the new ly synth esized
proteins w ere tested for their collagenase sen sitiv ity . In each
of the 3 experim en ts rep resen ted in Table 5, the c e ll-fr e e
incubation m ixture was divided after a 30 m inute incubation
period. One portion was further incubated with collagen ase,
the control with buffer, and both w ere assa^/edfor radioactivity
incorporated into r elea sed soluble p rotein s. The collagen ase
solution had been purified to rem ove alm ost com pletely any
i n on -collagen p rotease activity (106). The CPM lo st in the
i
collagen ase treated sam ple w ere considered to be from digested
j
collagen (37, 78-79). The experim en ts in Table 5 indicated
that about 8% of the proteins syn th esized by m edium p olysom es
| w ere collagen, and 50% of the proteins syn th esized by heavy
| p olysom es w ere collagen. The two polysom e fractions w ere about
l
equal in th eir rates of total protein syn th esis sin ce the incubation
sy stem with m edium polysom es had a 50% greater concentration
of p o ly so m es. By com parison, other stud ies have reported
c e ll-fr e e sy stem s producing 10-15% collagen (77-79) and 25-30%
| collagen (37). Since collagen was 50% of the total proteins
syn th esized , the heavy polysom e fraction was considered to have
I
an equivalent amount of co lla g en -sp ecific p o ly so m es, and was
used for subsequent studies.
78
Figure 16. A bsorbance Spectrum of Isolated P olysom e
F raction. F ractions of Heavy and Medium polysom es w ere
prepared as described in Chapter H. A sam ple of each fraction
was diluted in buffer and analyzed on a Beckm an
j spectrophotom eter. Each polysom e fraction has an A2 6 0 M 2 8 O
of 1. 77, indicating that they w ere reasonably free from
contam ination.
79
ABSORBANCE
0.9
a t
0.7
as
0.4
aa
0.1
ato 300 a40 140 310 lao
WAVELENGTH (nm)
80
C ollagenase
Table 5
Studies on Newly Synthesized P roteins
Incubation
System Treatm ent CPM % L oss
H P oly so m es
H P oly so m es
Buffer
C ollagenase
17, 586
8, 729 50. 4
M P olysom es
M P olysom es
Buffer
CollagBnase
28,416
27, 165 4 .4
M P olysom es
M P olysom es
Buffer
C ollagenase
30,813
27, 165 11. 7 ,
8 .1
Heavy and m edium p olysom es w ere prepared from skin and
incubated in a protein syn th esis system as d escrib ed in Chapter II. j
Although the absorbance spectrum of the heavy polysom e
fraction (F igure 16) did not indicate any contam inating m a terial,
it was considered p o ssib le that th ese heavy polysom es m ight
have been aggregates of sm a ller p oly so m es, bound together by
the in teraction of nascent collagen chains. T herefore equal
volum es of heavy polysom e solution w ere incubated for 30
m inutes at 37°C with collagen ase or with buffer. Both sam ples
w ere subsequently analyzed on su cro se gradients.
f
| A significant portion of the heavy p olysom es w ere found
i
i d isp ersed , sedim enting towards the top of the gradient. Figure
I
! 17 show s, how ever, that the sam e effect was observed with the
I
l
| control sam ple. Since th ere was no difference betw een the
| effect of the collagen ase and the control treatm en ts, it was
j assu m ed that d isp ersion of heavy p olysom es was the resu lt of
d isso cia tio n of rib osom es from the m essen g er RNA, and this
action could have m asked the potential action of the collagen ase.
The experim ent was repeated at 4 ° c , sin ce at th is tem perature
the degree of ribosom al d issociation was expected to dim inish.
C ollagenase was expected to be s till a ctive at this tem perature
| sin ce studies on th is enzym e indicated no significant drop in
■ activity at low ered tem peratures down to 8° or 9° C (13Q 131).
A s can be seen in F igure 17, the degree of d isp ersion was
significan tly reduced at the low er tem perature. Again th ere was
no difference betw een the collagen ase and the control sam p les. It
was concluded that collagenase its e lf had no detectable effect in
disp ersin g heavy p oly so m es. It is m ost lik ely that the heavy
p olysom es w ere not totally stable in the ab sen ce of the norm al
com ponents supporting protein sy n th esis, and that ribosom es
becam e d isso cia ted from the mRNA which th erefore sedim ented j
nearer the top of the gradient. Sim ilar d issociation of ribosom es
has been observed in other studies (73, 129), and a ribosom e
d issociation factor has been iso la ted from p olysom es (125). j
Although the polysom al mRNA m ay have becom e p artially stripped, j
j such mRNA retains its in tegrity and rem ains functional when
!
| added to a com plete protein syn th esis sy stem (96, 132). The data ;
i !
I
in Table 5 are evidence of the ability of the heavy polysom es to j
j
function when incubated with a ll the com ponents needed for
I
protein syn th esis.
The la rg e siz e of the heavy p o ly so m es, and their high rate
of collagen production indicated that they m ight contain
p olycistron ic m essen g er RNA, that is , a m essen g er RNA of
!
j sufficient length to tran slate sequentially a ll th ree p ro- C L chains
that form the trip le stranded collagen m o lecu le. This
hypothesis could be tested by adding an inhibitor to block
i 83
F igure 17. C ollagenase Treatm ent of Heavy P o ly so m es.
Heavy polysom es from Chick skin sedim enting in fractions 1-12
of the gradient w ere pooled and prepared as described in
Chapter II. 0. 67 m g of this fraction was incubated with
collagen ase at a fin al concentration of 0. 4 m g /m l. A control
sam ple was incubated with buffer. A fter a 30 m inute incubation,
the sam ples w ere analyzed on 34 m l 15-60% su cro se gradients.
The polysom es a re not a ll stable sin ce a considerab le fraction
has d issociated into sm a ller polysom es with a concom ittant
r e le a se of m onosom es and ribosom al sub-units (fractions 32-36).
T his effect is m ore pronounced at 37° than at 4° C. There is no
significan t difference in the d issociation effect betw een polysom es
treated with collagen ase and the controls.
84
FRACTION
ABSORBANCE (2 5 4 nm)
U
> 4
Control
re-in itia tio n of new rib osom es on the mRNA and then m easuring
the incorporation of a pulsed label into the term inal translation
product. To block re-in itia tio n , ATA was added sin ce it attaches
to the 40S ribosom al sub-unit and prevents it from attaching to the
mRNA (111). In con seq u en ce, only rib osom es already on the
p olysom e can continue to m ove along the m essen g er RNA and
syn th esize proteins (103). Once the la st ribosom e has been
r elea sed at the term inal codon, protein syn th esis stops. Thus,
i
the tim e of tran slation for the whole mRNA can be determ ined by
m easuring the appearance of released radioactive protein with
t
tim e. When protein syn th esis stops, the mRNA has been
translated, and the amount of labelled protein in solution i
t
I reach es a plateau le v e l.
! j
I Skin p olysom es w ere fir s t tested for th eir ability to syn th esize |
| i
I i
and r e le a se proteins for a tim e period sufficient to perm it
m easu rem en t of the tran slation tim e. F igure 18 shows that
protein syn th esis and r e le a se continued over the 40 m inutes of
incubation.
The tran slation tim es for both heavy p olysom es and m edium
!
{ p olysom es from sk in w ere then determ ined. Incubations w ere
1 ca rried out in the absence of initiation fa cto rs, and a lso in the
< i
p resen ce of ATA. T hese experim ents are shown in F igu res 19
| and 20, and sum m arized in Table 6. The translation tim e
F igure 18. The Synthesis and R elease of P roteins on P olysom es
from Chick Skin. 0. 7 m g of polysom es w ere incubated in a total
volum e of 1 m l, and 50 ul sam p les w ere taken over the 40
m in utes of incubation. The continuous in crea se in the amount
of r elea sed protein in d icates that, at the concentrations used
in th ese stu d ies, no factor has becom e lim iting, although the
rate of protein syn th esis d ecrea ses after 15 m in utes.
TIME (min.')
3H PROLINE INCORPORATED C P M lO " 2
— M W » (ft 0>
00
00
F igure 19. The T ranslation T im e of the M edium P olysom e
F raction Prepared from Chick Skin. Incubation was perform ed
as d escrib ed in Chapter II. In F ig. 20 A, re-in itia tio n was
blocked by the o m issio n of IF, and in F ig . 20 B re-in itia tio n
was blocked by the addition of AT A (0.1 mM). The translation
tim e is the tim e at which the le v el of r elea sed protein reach es
a plateau.
89
3h p r o l i n e i n c o r p o r a t e d
1
v O
o
CPM -10"2
F igure 20. The T ranslation T im e of the Heavy P olysom e
F raction from Chick Skin. Incubation was perform ed as
describ ed in Chapter II. In F ig. 21 A re-in itiation was
blocked by the o m issio n of IF , and sam ple volum es w ere 20 ul.
In F ig. 21 B re-in itiation was blocked by the addition of ATA
(0.1 mM), and sam ple volum es w ere 50 jil.
91
PROLINE INCORPORATED CPM
A
jo o-
160-
•0
40 -
X
CO
2000
1600
1200
•00
400
B
I ---------1— i i — i— i »■■■ ■ i---------r — i-------- 1 i »■ ■ i i
2 4 6 • 1 0 1 2 1 4 1 6 It 20 22 24 26 2« 30
T I M E ( m l a . )
92
Table 6
T ranslation T im e of P olysom es
P olysom e
F raction
M
M
H
H
H
A
93
Incubation
Condition
C om plete - IF
Com plete + ATA
T ranslation Time
______(m in .)
5 .6 t 1
Com plete - IF
Com plete - IF
+ ATA
C om plete + ATA
13. 0
15.6
12.0
) 13.5+ 1.5
for m edium p olysom es w as 5. 6 - 1 m in utes, and for heavy
p olysom es 13. 5 - 1. 5 m in u tes. T hese tran slation tim es are
proportional to the s iz e s of the two polysom e fraction s tested .
The m uch longer tran slation tim e of the heavy p olysom es a lso
in d icates that th ese p olysom es w ere com posed of m uch longer
in tegral mRNA m o lecu les than the m edium p o ly so m es, as vould
be req uired for them to be p olycistron ic.
To com pare the tran slation tim e for the heavy collagen
p oly so m es to valu es for other protein syn th esis sy ste m s, it was
n e c e ssa r y to convert th is value to the translation rate in amino
I acids syn th esized per m inute. The number of am ino acids in
j chick skin pro-CL chains was estim ated as follow s:
j chick skin tropocollagenGL chains contain 996 amino a cid s (13),
j and th ere are about 137 am ino acid s in the 'reg istra tio n peptide1
I
(24, th is is an average o f 3 valu es for chick em bryo p ro- C L
chains), resulting in a total of 1133 am ino acid s for the whole
pro-CL chain. A p olycistron ic mRNA coding for 3 pre-CL chains
would thus syn th esize 3399 am ino acids in one com plete round of
translation . T h erefore, using this estim ate for the siz e of the
i
p ro- CL chain, and assu m in g that th ese heavy collagen polysom es
w ere p o ly cistro n ic, th eir translation rate w as determ ined as about
255 am ino a cid s /m in u te. Table 7 lis ts published valu es for the
Table 7
T ranslation Rates for D ifferent M olecules
System M olecule a a /m in Source
E. coli trp operon
proteins 415 M orse, Baker & Yanofsky (133)
Hen oviduct, ovalbum in 69 P alm iter & Schimke (134)
tissu e s lic e conalbumin 85
R eticulocyte,
c e ll-fr e e
hem oglobin
ovalbum in
45
50
P alm iter (99)
Rat calvaria,
tissu e s lic e
collagenCLi
collagenC(»2
210
Vuust & P iez (82)
Rat calvaria,
tissu e s lic e
collagenC ll
collagenC l2
253 \
227 / 240
Vuust & P iez (84)
Chick skin,
c e ll-fr e e
collagenCCj + 2 255 t 30
-o
in
rate of syn th esis of collagen and other m o le c u le s. It can be seen
that the rate of syn th esis for the heavy collagen polysom es is in very
good agreem ent with valu es for collagen sy n th esis reported by Vuust
and P iez (82, 84). It is a lso apparent that la rg er polysom es have
a f a s t e r r a t e o f syn th esis than sm a lle r on es. This is probably due
i
j to the fact that the initiation step and p o ssib ly the r e le a se of the
protein are the ra te-lim itin g fa cto rs (99, 121, 133). Thus for
i
sm a ller p oly so m es, initiation and r e le a se would account for a
greater part of the total tran slation tim e,
j Having determ ined the tran slation tim e for the heavy collagen
p o ly so m es, an experim ent was done w herein the term in al tran s-
; lation product was p u lse-la b elled . R e-in itiation was b lock ed , and !
3 '
| a p u lse of H proline was added 9 m inutes after the start of |
incubation (9 m inutes = 2 /3 of the translation tim e). T herefore, |
I I
j only the protein that was syn th esized on the term in al third of the |
j m essen g er RNA could incorporate radioactivity. Since a
I
p o ly c is tr o n ic c o lla g e n mRNA w a s e x p e c te d to c o d e fo r two
pro-CLj c h a in s and o n e p ro -C tg c h a in , th e la s t th ir d o f th e
I m e s s e n g e r w ou ld co d e fo r e ith e r p r o - CCj o r p r o - C(*2 » and th u s
i
i only one of th ese two sp e cie s of CC chain could be radioactively
!
lab elled . By fractionating the products on acrylam ide g e ls, the
distribution of radioactivity incorporated into collagen was
!
{ m easured .
1 . 96
T able 8
A nalysis of P u lse -la b e lle d C ollagen on A crylam ide G els
Experim ent
1.
H P rolin e Incorporated
CPM
0
433
71
2*.
32
10
C L 2
36
258
2. 2189
1390
17
156
1130
188
3. Control 4606 1314 1282
A crylam ide g els P j - P^ w ere la yered with sam ples of
collagen syn th esized on the term in al third o f the polysom al
mRNA. Incubations w ere perform ed as d escrib ed in Chapter II.
97
The experim ent was perform ed as d escrib ed in Chapter IL
In addition, a control incubation w as run under norm al conditions,
without any inhibitors. T able 8 show s the resu lts of th ese
exp erim en ts. The gel with the control sam ple shows that the bulk
of the rad ioactivity is found in the collagen Q band, and both the C L *
and C L 2 collagen bands a re w ell lab elled . G els from the p u lse-
lab elled sam p les show that the bulk o f the la b e l was again found in
the Q collagen band. The CLl band contained very little radio
activity in any o f the four g e ls, w hile the CL2 band was
| i
substan tially lab elled in two of the four g e ls. The fact that the j
! t
I j
collagen bands in the four p u lse-la b elled g e ls do not show uniform
: incorporation of la b el is probably due to the technical d ifficu lties j
i j
j encountered in preparing the sam p les for electro p h o resis. A fter !
!
the incubation sy stem had been stopped, it w as n ecessa ry to iso la te j
and concentrate the new ly syn th esised collagen to fa cilita te an a ly sis
on acrylam ide g e ls. The p olysom es w ere rem oved by high speed
centrifugation, but the enzym es rem ained in the supernate with the
collagen. C ollagen for g e l sam p les Pj and P 2 was iso la ted by
sodium chloride p recip itation at pH 3. 5. When the sam ple was
resuspended in buffer it did not to ta lly d isso lv e, and so m e of the
radioactive collagen m ay have b een trapped in the insolu ble
fraction. The procedure was m odified for sam ples P 3 and P^, |
A fter pelletin g the polysom es from the sam p le, tl e supernate
was adjusted to a pH of 4. 0 to p recip itate the contam inating enzym es.
T his precip itate w as rem oved by pelletin g, and the collagen was
; iso la ted as before. Again a ll of the precip itate did not d isso lv e,
but it can be seen for gels P 3 and P4 (Table 8 ) that m ore lab elled
collagen m igrated on the g e ls.
The data ex p ressed in T able 8 are considered to be
sign ifican t. It m ust be str e sse d that th ey do not prove
co n clu sively that the collagen mRNA is p o ly cistro n ic. H ow ever,
the control gel shows that Cbj chains w ere substantially lab elled
under norm al conditions, and that they could m igrate on the gel
under the procedural conditions em ployed. This in d icates that the
i
| C L j chains w ere not, som ehow , se le c tiv e ly entrapped by the
insolu ble protein fraction in the sam ple. Thus, the fact that the
CLj band had very little radioactivity in a ll 4 gels with p u lse -
lab elled sa m p les, and that on ly the CL 2 band could be su b
stan tially lab elled certainly su ggests that CL3 was the term in al
tran slation product. The appearance of the m ajority of the
rad ioactivity in collagen is in agreem ent with a recen t study
which showed the form ation of disulfide linked pro-CL chains
within a few m inutes following a four m inute p u lse (24).
CHAPTER IV
SUMMARY
The an alysis of p olysom es from chick em bryo sk in and from
chick em bryo wings and le g s dem onstrated that th ese tissu e s
contain a wide spectrum of polysom es having sedim entation
constants up to 2000 S or greater. A heavy polysom e fraction in
skin that was m em brane-bound w as solub ilized intact with
I
i
detergen ts. This heavy polysom e fraction had a sedim entation
i
constant of about 1600 S and was estim ated to contain about 116
i
i
| rib osom es per mRNA.
P olysom es from both tissu e so u rces w ere able to stim ulate the
incorporation of tritiated proline into proteins in a c e ll-fr e e system
under appropriate conditions. When the conditions for protein
syn th esis w ere optim ized, the two sy stem s w ere equally
I
efficien t in th eir rate of protein sy n th esis.
The two c e ll-fr e e sy stem s w ere sign ifican tly different in th eir
I dependance on factors required for the support of p rotein sy n th esis.
[
! Skin polysom es w ere m ost effective at a Mg’ * " * ” concentration of
I
4. 5 m M , and a K+ concentration o f 90 mM , while the wing and leg
I
i
! ioo
J p olysom es functioned b est with Mg'1 "1 * at 7. 5 mM and K+ at 150 mM.
t
The ionic ratio for the two sy stem s was the sam e. The two c e ll-
fr e e sy stem s a lso differed in th eir resp on se to various preparations
of soluble factors and initiation fa cto rs. Skin p olysom es w ere
inhibited by a pH 5 enzym e fraction, and w ere stim ulated by an
AS7 Q enzym e fraction. Wing and leg p olysom es w ere stim ulated
m od erately by pH 5 en zym es, and substantially stim ulated by
ASyg enzym es although at v ery high concentrations the AS7 0
enzym es w ere le s s effectiv e.
Both polysom e sy stem s responded dram atically to the addition
|
, of initiation fa cto rs, but at the highest concentration initiation :
i i
fa cto rs from skin w ere le s s than optim um , indicating the p o ssib le j
i p resen ce of an inhibitory factor. The various inhibitory effects
| 1
i ;
seen with the different enzym e or factor preparations could be ex - 1
i
I
plained by the p resen ce of a sin gle inhibitory factor in each sy stem , j
The two inhibitory factors had different p h y sica l-ch em ica l
p ro p erties, indicated by the different conditions under which they
rem ained soluble or w ere precip itated. The inhibitor from skin
precip itated at a pH of 5. 2, and with am m onium sulfate at 40% of
j l
| saturation. It m ay a lso be a sso cia ted with the polysom al p ellet. |
j The inhibitor from w ings and le g s was soluble at pH 5. 2, but |
precip itated betw een 40% and 70% of saturation with am m onium su l- j
; fa te. It could not be detected in the polysom al p e lle t. j
Skin p olysom es w ere separated into heavy and m edium fraction s.
A nalysis by absorbance sp ectroscop y indicated that such polysom e
preparations contained no detectable contam ination. Skin
p olysom es w ere able to m aintain the syn th esis and rele a se of
proteins for at le a s t 40 m inutes when incubated under standard
conditions. A collagen ase a ssa y of newly syn th esized proteins
dem onstrated that 8 % of the proteins syn th esized by m edium
p olysom es from skin was collagen , and 50% o f the proteins
synth esized by heavy skin p o lysom es was collagen.
I
The stab ility of the heavy p olysom es was exam ined by
i
j incubation with buffer or co lla g en a se, follow ed by su crose
: gradient sedim entation. T here was no sp ecific affect due to
1 collagen ase, but a portion o f the p olysom es w ere d isp ersed and
I
sedim ented toward the top of the gradient, m o st lik ely due to
d issociation of ribosom es from the mRNA. Such ribosom al
d issociation has been reported in other stu d ies. The degree of
d issociation was m ore sign ifican t at 37° than at 4° C.
The tran slation tim e o f the m edium and heavy polysom e
fractions from skin was m easu red under conditions where r e -
I
initiation was blocked, preventing new rib osom es from attaching
at the 5 1 end of the mRNA. Under th ese conditions the ribosom es
; alread y on the polysom e could fin ish their p a ssa g e along the mRNA
! 102
and r e le a se their com pleted protein chains. The*time at which
the amount of r ele a sed ra d io -a ctiv e protein in solution reached a
plateau was con sid ered to be the tim e required to com plete o n e
round o f translation on the m essen g er RNA. It was determ ined
by th ese experim en ts that the translation tim e for m edium
p olysom es was 5. 6 * 1 m in utes, and for heavy polysom es it was
13. 5 * 1. 5 m in u tes. T hese translation tim es w ere proportional
to the s iz e s of the mRNA in the polysom e fraction s.
F rom values in the litera tu re it was estim ated that a collagen
| pro-CC chain contains 1133 am ino a cid s. A ssum ing that the
! heavy collagen p o lysom es w ere p o lycistron ic, the translation tim e
j was equivalent to a protein syn th esis rate of 255 amino acids per
i minute for th ese collagen p o ly so m es. This value is in good
i
| agreem ent with published data on the rates of collagen syn th esis.
B ecau se of the s iz e of the heavy p oly so m es, their efficien cy
in synthesizing collagen , and the length of th eir translation tim e,
it was proposed that th ese p olysom es contain polycistron ic
collagen mRNA coding for the sequential translation of the 3
CL chains that constitute the trip le stranded collagen m olecu le.
i This w as tested by pu lse lab ellin g the collagen synthesized on the
|
! term inal third of the mRNA; the pu lse lab elled collagen was
; analyzed on acrylam id e g e ls. A sam ple from a control
incubation showed that both CL} and C l 2 chains w ere substantially
lab elled . In sam p les from p u lse-la b elled incubations, the C lj
band had no significant radioactivity incorporated in any of 4 g e ls.
The Cl 2 band in 2 of the 4 g els had incorporated a substantial
amount of tritiated p rolin e. From the distribution of lab elled
collagen on the control gel and the 4 pu lse -la b elled g els it
appeared that the C l£ chain was the term inal tran slation product.
In a recen t review Davidson and B ritten stated that no evidence
ex isted for a p olycistron ic mRNA in a eukaryotic organism (134).
They did consider that p olycistron ic HnRNA m ight ex ist in
eukaryotic organ ism s. Though the above experim en ts do not
i
; afford con clu sive proof of the existen ce of a polycistron ic mRNA,
| i
i i
j the data a re considered to be significan t. The evidence from the i
i i
various experim ents with the heavy polysom e fraction dem onstrated: \
1) the heavy polysom e fraction contained a la rg e amount of collagen
t
mRNA sin ce over 50% of the proteins syn th esized w ere collagen;
2 ) the collagen polysom es a re la rg e enough to be polycistronic;
3) th eir translation tim e is long enough for a p olycistron ic mRNA;
4) based on the assum ption that th ese polysom es are p o ly cistro n ic,
1 the tran slation rate for the syn th esis of collagen is in good a g r ee -
j
1 merit with published valu es; 5) the CI2 chain appeared to be the
i
; term inal translation product. Thus the weight o f the evidence
»
I 104
from th ese experim ents certain ly su ggests that the heavy
polysom e fraction from chick em bryo skin contains polycistron ic
collagen m essen g er RNA.
105
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Creator Traut, Thomas Wolfgang (author)

Core Title Studies On The Cell-Free Synthesis Of Collagen On Polysomes From Chick Embryo Connective Tissues

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Cellular and Molecular Biology

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Advisor Petruska, John A. (committee chair), Dunn, Arnold S. (committee member), Nimni, Marcel E. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-743377

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