The effect of chloramphenicol on the in vitro synthesis of 14C-proline peptides as directed by
polycytidylic acid
by James Duard Irvin
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by James Duard Irvin (1970)
Abstract:
A method is presented which partially separates a homologous series of proline: peptides by the
technique of peptide mapping. This technique was applied to the analysis of the 14 C-proline peptides
synthesized in vitro as directed by polycytidylic acid and the effect of chloramphenicol on. the. peptide
synthesis.. The incorporation system was characterized and it was found that it was a polysomal
system. The peptide products were determined and found to consist of very small peptides (di- and.
tripeptides) and very large peptides of undetermined molecular weight as major products.
Chloramphenicol was found to inhibit the formation of both major species of peptides at low
concentrations. (I to 25 μg/ml). The synthesis of the large peptides was found to be more sensitive to
chloramphenicol than that of the small peptides.
The results from this study indicate that chloramphenicol acts at some step early in protein synthesis
after the formation of the initiation complex. The data allow the exclusion of various known steps
in.protein synthesis as the site of the antibiotic's action, namely, translocase activity, ribosomal
movement and the explusion of deacylated t-RNA. It is postulated that chloramphenicol's action occurs
at the donor (peptidyl) site of the ribosome. THE EFFECT OF CHLORAMPHENICOL ON THE IN VITRO SYNTHESIS OF
14C-PR O LIN e PEPTIDES AS DIRECTED BY POLYCYTIDYLIC ACID
by
JAMES DUARD IRVIN
A t h e s i s s u b m itte d to th e G ra d u a te F a c u lty in p a r tia l
fu lfillm e n t o f th e re q u ire m e n ts fo r th e d e g re e
of
DOCTOR OF PHILOSOPHY
■in
C h e m istry
MONTANA STATE UNIVERSITY
B o zem an , M o n ta n a
M a r c h , 19.70
ii i
ACKNOWLEDGEMENTS
The a u th o r th a n k s :
L . JEAN HAINES- IRVIN, e s p e c i a l l y , fo r h e r u n d e rs ta n d in g an d
e m o tio n a l s u p p o rt th ro u g h o u t th is s tu d y .
M R. a n d MRS. D . J . IRVIN fo r th e ir c o n fid e n c e a n d u n d e r s ta n d in g .
DR. GORDON RAY JULIAN fo r h is c r e a tiv e g u id a n c e a n d c o n s tr u c t­
iv e c r itic is m o f th is s tu d y .
DR. SAMUEL JOHN ROGERS, fo r h is i n t e r e s t a n d a d v ic e .
DR. HELEN J-. CAMERON fo r h e r h e lp fu l d i s c u s s i o n .
MISS KATHY CARD for te c h n ic a l a s s i s t a n c e .
The N a tio n a l D e fe n s e a n d E d u c a tio n Act a d m in is tra tio n fo r fin a n ­
c i a l s u p p o rt (f e llo w s h ip num ber 6 7 -0 6 6 0 3 ).
TABLE OF CONTENTS
CHAPTER
I.
PAGE
IN T R O D U C T IO N ............. .............................................................................. .
I
C h lo r a m p h e n ic o l............................................................................................
I
P o ly C -D ir e c te d
C -P ro lin e I n c o r p o r a t i o n ....................................
3
.........................................................................................................
4
MATERIALS AND METHODS . .....................................................................
7
B u f f e r s ...............................................................................................................
7
G l a s s - D i s t i l l e d W a t e r ...............................................................................
7
P re p a ra tio n o f S o lu tio n B e n t o n i t e ........................................................
8
G la s s - D is tille d . H y d ro c h lo ric A c i d ....................................................
8
R ib o n u c le a s e R em oval ...........................................................
9
R ibosom e P re p a ra tio n ...............................................................; . . . . .
9
R a tio n a le
.II.
14
■
- DEAE. C olum n ! .P r e p a r a tio n ................
11
S-IOO C o n c e n tra tio n . . . . .................................................................
11
P h en o l E x tra c tio n o f P o ly c y tid y lic A c i d ...........................................
12
In c o rp o ra tio n P ro c e d u re ............................................................................
12
P la tin g P ro c e d u re fo r th e
14
C -P ro lin e P e p tid e s
...................
- P a r tia l A c id .H y d ro ly z a te o f P o l y p r o l i n e ...........................................
P re p a ra tio n o f th e
14
C -P ro lin e P e p tid e s for A n a l y s i s ................
13
14
14
C o m b in ed E le c tro p h o re s is -C h ro m a to g ra p h y o f th e
P ro lin e P e p tid e s ......................................................................................
R a d io a u to g ra p h P ro c e d u re .......................................
14
15
V
CHAPTER
II.
PAGE
(C o n tin u e d )
Q u a n tita tiv e D e term in atio n , o f th e S e p a ra te d
^ C - P r o l i n e P e p t i d e s .............................
. ..1 6
S u c ro s e D e n s ity G ra d ie n t A n a l y s i s ........................................................ 16
, II I .
EXPERIMENTAL,RESULTS .................................
18
.I V .
D IS C U S S IO N .............................................
37
LITERATURE CITED
......................................................................................................... 49
■TABLE OF FIGURES
FIGURE
PAGE
1 . E le c tro p h o re s is o f P ro lin e a n d P ro lin e P e p t i d e s .................................
21
2 . P a p e r C h ro m a to g ra p h y o f P ro lin e a n d P ro lin e P e p t i d e s ....................
22
3 . C o m b in ed E le c tro p h o re s is -C h ro m a to g ra p h y o f a P a r tia l
Acid. H y d ro ly s a te o f P o l y p r o l i n e ..........................................
23
4 . The E ffe c t o f M a g n e s iu m ,Io n C o n c e n tra tio n on th e In c o r p o ra tio n o f
14
C - P ro lin e in to P r e c ip ita b le P e p t i d e s ........................... 24
5 . Thd E ffe c t o f P o ly c y tid y lic Acid. C o n c e n tra tio n o n th e
In c o rp o ra tio n o f
14
C -P ro lin e : in to P r e c ip ita b le P e p tid e s . . . . .
25
6 . A u to ra d io g ra m .o f th e S e p a ra te d ^ C - P r o l i n e P e p tid e s
S y n th e s iz e d .in v itro a s D ire c te d b y P o l y C .................................... .
26
7 . A u to rad io g ram o f th e 15% TCA. P r e c ip ita b le P e p tid e
A n aly sed , b y C o m b in e d v E le c tro p h o re sis -C h ro m a to g ra p h y
. . . .
27
...........................................................
28
8 . A u to rad io g ram o f th e 15% TCA, P r e c ip ita te S u b je c te d .to
. P a rtia l A cid H y d ro ly s is a s A n a ly z e d b y C om bined
• E le c tro p h o re s is -C h ro m a to g ra p h y
9 . S u c ro s e D e n s ity G ra d ie n t A n a ly s is o f a S ta n d a rd
. I n c o rp o ra tio n .(250 n l)
..............................................................
29
3
I. 0 . S u c ro s e D e n s ity G ra d ie n t A n a ly s is o f th e H -P o ly C rib o so m e C o m p lex In c u b a te d w ith ■P u rifie d R i b o s o m e s ................
II.
30
The E ffe c t o f C h lo ra m p h e n ic o l o n .th e In c o rp o ra tio n o f
^ C - P r o l i n e in to P r e c ip ita b le P e p t i d e s ......................................................31
. vii
FIGURE
PAGE
1 2. The E ffe c t o f C h lo ra m p h e n ic o l o n th e D is trib u tio n o f
^ C - P r o l i n e P e p tid e s (I ( ig / m l) .................................... .............................
32
1 3. The E ffe c t o f C h lo ra m p h e n ic o l o n th e D is trib u tio n o f
^ C - P r o lin e : P e p tid e s (5 iag/ml) ..................................................................
33
1 4. The E ffe c t o f C h lo ra m p h e n ic o l o n th e D is trib u tio n o f
^ C - P r o l i n e P e p tid e s ( I O n g /m l) .............................
34
1 5. The E ffe c t o f C h lo ra m p h e n ic o l on th e D is trib u tio n o f
^ C - P r o l i n e P e p tid e s ; (25 p g / m l ) ..............................................................
35
1.6. The E ffe c t o f C h lo ra m p h e n ic o l on. P ro lin e P e p tid e
S y n th e s is
..........................
36
. viii
Ab s t r a c t
A m e th o d .is p r e s e n te d w hich, p a r tia lly s e p a r a te s a h o m o lo g o u s
s e r i e s o f p ro lin e : p e p tid e s, b y th e te c h n iq u e o f p e p tid e m a p p in g „ T his
te c h n iq u e w a s a p p lie d to th e a n a ly s is ' o f th e ^ C -p r o lin e p e p tid e s s y n ­
t h e s i z e d in v itro a s d ir e c te d b y p o ly c y tid y lic a c id a n d th e e f f e c t o f
c h lo ra m p h e n ic o l on. the. p e p tid e s y n t h e s i s .. The in c o rp o ra tio n s y s te m w a s
c h a r a c te r iz e d a n d .it w a s found th a t i t w a s a p o ly s o m a l s y s te m . The
p e p tid e p ro d u c ts w e re d e te rm in e d , an d . fo u n d to c o n s is t o f v e ry s m a ll
p e p tid e s ( d i- and. tr ip e p tid e s ) an d v e ry la rg e p e p tid e s o f u n d e te rm in e d
m o le c u la r w e ig h t a s m a jo r p r o d u c ts .
C h lo ra m p h e n ic o l w a s foun d to. in h ib it th e fo rm atio n , o f both, m ajo r
s p e c ie s o f p e p tid e s a t lo w c o n c e n tra tio n s . ( I to 25 p g / m l) . The s y n th e s is
o f th e la rg e p e p tid e s w a s .found to b e m ore s e n s itiv e to c h lo ra m p h e n ic o l
th a n th a t o f th e s m a ll p e p t i d e s .
The r e s u lts from th is s tu d y in d ic a te th a t c h lo ra m p h e n ic o l a c ts a t
so m e s te p e a r ly in. p ro te in s y n th e s is a f te r th e fo rm atio n o f th e in itia tio n
c o m p le x . The d a ta a llo w th e e x c lu s io n o f v a rio u s know n s te p s , in .p ro ­
te in s y n th e s is a s th e s i t e o f th e a n t i b i o t i c 's a c t i o n , n a m e ly , tr a n s lo c a s e a c t i v i t y , rib o s o m a l m o v e m e n t, a n d th e e x p lu s io n o f d e a c y la te d
t-R N A . I t i s p o s tu la te d th a t c h lo ra m p h e n ic o l's a ctio n , o c cu rs, a t th e
d o n o r (p e p tid y l) s i t e o f th e rib o s o m e .
CHAPTER. I
INTRODUCTION
C h lo ra m p h e n ic o l
C h lo ra m p h e n ic o l (CAP) i s a p a n tib io tic w h ic h h a s b e e n sh o w n .to
in h ib it p ro te in s y n th e s is in b a c te r ia (1 ,2 , 3 ).
It h a s b e e n s h o w n .th a t
CAP d o e s n o t in h ib it am in o a c id a c tiv a tio n (4) n o r d o e s i t in h ib it th e
fo rm a tio n o f a m in o a c y l-t-R N A ..(5 ). CAP in h ib its am in o a c id p o ly m e riz a ­
tio n in c e l l - f r e e " in c o rp o ra tio n s y s te m s " (6,7) a n d th is in h ib itio n is
s t e r e o s p e c i f i c , b e in g o b s e rv e d o n ly w ith th e D (-) th re o s te r e o is o m e r
( 8) .
S tu d ies, o n rib o s o m a l c e ll - f r e e am in o a c id p o ly m e riz in g s y s te m s
le a d s e v e r a l a u th o rs to a s s u m e th a t G A P's a c tio n o c c u r s o n th e rib o so m e
(8 ,9 ,1 0 ). T h is s u p p o s itio n w a s s u p p o rte d b y s u b s e q u e n t w ork re p o rtin g
th a t r a d io a c tiv e CAP b in d s to th e 508 s u b u n it o f th e rib o so m e (11,12).
A lso th is b in d in g w a s show n, to. b e s te r e o s p e c if ic . (13,14) a n d o c c u rs in a
1:1 e q u iv a le n c e b e tw e e n . CAP an d th e rib o so m e. (15). At h ig h e r c o n c e n ­
tr a tio n s o f th e a n tib io tic a d d itio n a l a m o u n ts b eco m e bound. (16).
It w a s s u g g e s te d th a t CAP in h ib ite d th e b in d in g o f m-RNA to th e
rib o so m e. (8 ). T his h y p o th e s is w a s t e s t e d w ith v a rio u s s y n th e tic m e s ­
s e n g e r s a n d no s ig n if ic a n t in h ib itio n o f b in d in g w a s o b s e r v e d (9,17,18).
The p o s s ib il ity th a t CAP in h ib ite d a tta c h m e n t o f am in o a c y l-t-R N A
to th e m e s s e n g e r -rib o s o m e c o m p le x w a s in v e s tig a te d b y v a rio u s a u th o r s ,
th e m a jo rity o f w h ic h re p o rte d no in h ib itio n of p h e n y la la n y l-t-RNA b in d in g
2
. to p o ly U -rib o s o m e c o m p le x e s (1 8,19,20), b u t o th e r a u th o rs re p o rte d lo w
le v e l (10-15%) in h ib itio n o f ly s y l-t-R N A b in d in g to p o lyA -rib o so m e co m ­
p le x e s (15), Y et o th e r a u th o rs re p o rt th a t CAP in h ib its th e b in d in g of
p h e n y ia la n y l-t-R N A to th e rib o so m e b u t n o t th e b in d in g o f ly s y l-t-R N A
o r p ro ly l-t-R N A .(21). R e c e n tly i t h a s b e e n sh o w n th a t CAP in h ib its th e
b in d in g o f th e am ino a c y l te rm in u s o f p h e n y la la n y l-t-R N A to th e 5 OS
s u b u n it o f th e rib o so m e (22) .
O ne s trik in g fe a tu re o f CAP'S a c tio n on the. in c o rp o ra tio n o f am ino
a c id s in to p e p tid e s in c e l l - f r e e s y s te m s is th a t its in h ib ito r y e ffe c t
v a r ie s w ith th e m-RNA u s e d . N a tu ra l m e s s e n g e r s a re m u ch m ore s e n s i ­
tiv e to C A P .than a re s y n th e tic m e s s e n g e r s . (9 ,2 3 ). A lso th e c o m p o sitio n
o f h e te ro p o ly m e r s y n th e tic m e s s e n g e r s is d ir e c tly r e la te d to th e ir s e n s i ­
tiv ity to CAP; th e m ore c y tid y lic a c id c o n ta in e d in th e p o ly m e r th e
g r e a te r is th e in h ib itio n o f am ino acid , in c o rp o ra tio n b y CAP. (9,17). In
. th e c a s e o f th e h o m o p o ly m eric s y n th e tic m e s s e n g e r s th e o rd e r of in h ib i­
tio n by CAP is polyC . > polyA > p olyU (1 7 ,2 4 ).
I n v e s tig a tio n o f th e to ta l p e p tid e p ro d u c ts from a polyA d ire c te d
:. ly sin e , in c o rp o ra tio n s y s te m in th e p r e s e n c e an d a b s e n c e o f CAP r e ­
v e a le d th a t CAP in h ib ite d th e fo rm a tio n o f la rg e r p e p tid e s (4-11 ly s in e
r e s id u e s ) b u t p e rm itte d a n d e v e n s tim u la te d th e fo rm a tio n o f d i - an d
tr i p e p t i d e s .(2 5 ). A n a ly s is o f th e .tr y p tic p e p tid e s o f th e c o m p le te d an d
. r e le a s e d c o a t p ro te in produced, b y th e n a tu ra l m-RNA d e riv e d from th e
3
b a c te rio p h a g e M S -2 sh o w e d an. o v e ra ll a n d e q u a l d e p r e s s io n o f a ll.th e
p e p tid e s in th e p r e s e n c e o f CAP (2 6 ,2 7 ).
The a n tib io tic purom ycin. in h ib its p ro te in s y n th e s is b y re a c tin g
w ith p e p tid y l-t-R N A to form p e p tid y lp u ro m y c in (2 8 ,2 9 ,3 0 ) . I t h a s b e e n
s u g g e s te d t h a t .t h i s r e a c tio n m ig h t s e rv e a s a m odel o f p e p tid e bond
s y n t h e s i s . E x te n s iv e s tu d ie s h a v e b e e n m ad e on th e e f f e c t o f CAP on
th e p u ro m y cin r e a c tio n i n . v iv o an d in v i t r o . M an y a u th o rs a rg u e th a t
th e in h ib itio n o f th e p u ro m y c in re a c tio n , b y CAP in d ic a te s th a t CAP s p e ­
c if ic a l ly in h ib its th e e n zy m e p e p tid y l tr a n s f e r a s e w h ic h c a ta ly z e s th e
fo rm a tio n o f th e p e p tid e bond (2 9 ,3 1 ,3 2 ,3 3 ). Y et o th e r a u th o rs in te rp re t
th e ir r e s u lts from s im ila r s tu d ie s .to m ean th a t CAP d o e s n o t in h ib it
p e p tid y l tr a n s f e r a s e b u t som e s te p p rio r to its a c tio n (3 4 ).
In su m m a ry , i t a p p e a rs th a t CAP'S s i t e o f a c tio n i s on. th e 508
s u b u n it o f th e rib o so m e a n d th a t i t s s te ro .s p e c ific a c tio n , o c c u rs a t or
b e fo re th e p e p tid y l tr a n s f e r a s e s te p o f .p ro tein s y n t h e s i s .
... PolyC D ire c te d
14
C -P ro lin e In c o rp o ra tio n
P olyC w a s f i r s t re p o rte d to d ir e c t ^ C - p r o l i n e in c o rp o ra tio n in to
p e p tid e s in a 8 -3 0 s y s te m (35) a f te r p re v io u s s tu d ie s in d ic a te d th a t it
w o u ld n o t a c t a s a s y n th e tic m e s s e n g e r (36)
The. p o ly C d ir e c te d in ­
c o rp o ra tio n o f p ro lin e w a s fu rth e r c h a r a c te r iz e d in a. rib o s o m a l c e l l - f r e e
s y s te m .(3 7,38) a n d p o ly C w a s show n to b in d to th e 308 s u b u n it o f th e
rib o so m e .(.38). T his m e s s e n g e r has- n o t been "u sed e x te n s iv e ly in s tu d ie s
4
o f c e l l - f r e e p ro te in s y n th e s is s in c e i t h a s n o t b e e n sh o w n , to prom ote
p rp lin e in c o rp o ra tio n n e a r ly a s w e ll a s p o iy U p ro m o tes p h e n y la la n in e
in c o rp o ra tio n o r polyA p ro m o tes ly s in e in c o r p o r a tio n . F o r ex am p le,, from
a r e c e n t s tu d y on sp a rso m y c in . a c t i o n , p o ly C d ir e c te d p ro lin e in c o rp o ra ­
tio n w a s o n ly o n e - tw e n tie th o f th a t o b s e r v e d fo r polyA d ir e c te d ly s in e
in c o rp o ra tio n (3 9 ). A lso i t h a s b e e n s h o w n .th a t p o ly C h a s a m uch lo w e r
a f f in ity fo r th e rib o so m e th a n po iy U (4 0 ). The o n ly m a jo r u s e o f th e
p o ly C -p ro lin e s y s te m h a s b e e n in s tu d ie s oh. th e e f f e c t o f v a rio u s, a n t i ­
b io tic s on c e l l - f r e e p ro te in s y n th e s is (3 9 ,4 1 ,4 2 ,4 3 ,4 4 ,4 5 ,4 6 ) „
R a tio n a le
The s tu d y o f-th e e f f e c t o f CAP on th e p o ly C s tim u la te d in c o rp o ra tio n o f
14
C -p ro lin e m vitro, is u n d e rta k e n in. an a tte m p t to d e te rm in e th e
m e c h a n ism o f a c tio n o f CAP o r a t l e a s t c o n trib u te to th e d e te rm in a tio n
I
. o f th e a n t i b i o t i c 's m e c h a n is m o f a c tio n .
CAP i s a w id e ly u se d , to o l in s tu d ie s o f th e b io c h e m ic a l p r o c e s s e s
o c c u rrin g in m ic ro o rg a n is m s . The d e te rm in a tio n o f e x a c tly how th e
a n tib io tic a f f e c ts p ro te in s y n th e s is w o u ld h e lp to m ak e i t s a p p lic a tio n
. b e tte r u n d e rs to o d and. p ro b a b ly le a d to i t s g r e a te r a p p lic a tio n a s a
b io c h e m ic a l t o o l . A lso o n c e th e a n t i b i o t i c 's E ffe c t o n p ro te in s y n th e s is
i s d e te rm in e d i t sh o u ld le ad , to a fu rth e r e lu c id a tio n o f so m e o f th e s te p s
in v o lv e d in ;p r o te in s y n t h e s i s .
5
O n ly tw o in v itr o p ro te in s y n th e s iz in g sy ste m s, h a v e b e e n s u b ­
j e c t e d to c o m p le te a n a ly s is o f th e p e p tid e p r o d u c ts . O ne is th e polyA
14
d ir e c te d in c o rp o ra tio n , o f . C - I y s in e in. w h ic h th e h o m o lo g o u s ly s in e
p e p tid e s w e re f r a c tio n a te d by c a rb o x y m e th y lc e llu lo s e co lu m n ch ro m a­
to g ra p h y (4 7 ,2 5 ). The o th e r is th e in. v itr o s y n th e s is o f M S-2 c o a t
p ro te in b y M S-2 RNA (2 7 ). R e s u lts from GA P's a c tio n o n th e polyA ly s in e s y s te m m ay be o b je c te d to on th e fo llo w in g g ro u n d s: (I) The
s y s te m p ro d u c e s p e p tid e s v a ry in g in s i z e from (Iy s)^ to ( I y s ) ^ an d so i t
w o u ld se e m th a t th is d o e s n o t a c tu a lly s im u la te v e ry c lo s e ly th e a c tu a l
c o n d itio n s o f in v iv o p ro te in s y n th e s is in w h ic h m uch la r g e r p o ly m ers
a re p ro d u c e d . (2) The fo rm atio n o f .the la rg e am o u n ts o f d i - a n d tr ily s in e
o b s e r v e d m ay be d ir e c te d by o lig o a d e n y la te s s y n th e s iz e d in s i t u , th u s
g iv in g r is e to the- p ro d u c tio n o f .th e s e p e p tid e s in fa r g r e a te r am o u n ts
th a n th o s e a c tu a lly p ro d u c e d by th e ad d ed ..lo n g c h a in polyA . In fa c t,, it
h a s b e e n sh o w n t h a t o lig o a d e n y la te s d ir e c t th e s y n th e s is , o f d i - and
.trily s in e (48) . The r e s u lts on th e p e p tid e a n a ly s is o f th e M S-2 s y ste m
a re a t p r e s e n t in c o m p le te .
The u s e o f th e p o ly C -p ro lin e s y s te m w o u ld c irc u m v e n t e rro n e o u s
r e s u lts a r is in g fro m .th e fo rm atio n o f o lig o c y tid y la te s s in c e CTP m ay be
e x c lu d e d from .the. in c o rp o ra tio n s y s te m . A lso th e s iz e o f th e p ro lin e
p e p tid e s s y n th e s iz e d in v itro w a s unknow n, p rio r to th is s tu d y an d a s
w ill b e sh o w n la t e r the. p o ly C -p ro lin e s y s te m p ro d u c e s a s o n e o f its
p ro d u c ts a. la rg e p o ly m e r o f p r o li n e .
6
F or th is s tu d y i t w a s n e c e s s a r y to d e v is e a m eth o d fo r th e s e p a r a ­
tio n o f a h o m o lo g o u s s e r i e s o f p ro lin e p e p tid e s , p re p a re a c e ll - f r e e
s y s te m w h ic h uses. p o ly C to s tim u la te th e fo rm atio n o f
14
C -p ro lin e p e p ­
tid e s i n y ie ld s s u f f ic ie n t fo r a n a ly s is b y th e s e p a ra tio n ; te c h n i q u e , a n d
a p p ly th e s e p a ra tio n , te c h n iq u e to a n a l y s i s o f th e p e p tid e s, p ro d u c e d in
the, in v itro s y s te m in th e p r e s e n c e a n d a b s e n c e o f CAP.
CHAPTER . II
MATERIALS AND METHODS
.. B uffers
BC
0.1 M am m onium b ic a r b o n a te (NH^HCOg); 0.016 M m ag n esiu m
a c e t a t e (MgACg); pH 7.95 u n a d ju s te d .
. T
0.02 M.. T ris(h y d ro x y m e th y l)a n iin o m e th a n e (T ris ); 0.08 M
ammonium, c h lo rid e (N H ^Cf)? 0.016 M M gAc^; pH a d ju s te d to
7.95 w ith g l a s s - d is tille d , (gd) h y d ro c h lo ric a c id (H C l).
LMBC
0.1 M NH4 H C O 3 ; 0.01 M M gA c2 ; pH 7.95 u n a d ju s te d .
HMBC
0.1 M N H 4 H C O 3 ; 0.1 M -M gA c2 ; pH 7.95 u n a d ju s te d .
OC
0.25 N H 4 C l; 0.005 M MgAc3 ; .0.01 M .T ris -H C l; pH 7.8.
OD
0.50 NH4 C l; 0.1 M MgAc2 ; 0.01 M T ris-H C l; pH 7.8.
N o te:
The p r e s e n c e o f a p lu s s ig n (+) in b u ffe r d e s ig n a tio n , e . g . BC+ ,
in d ic a te s th a t- th e b u ffe r h a s b e e n m ade. 0.006 M in
2 - m e r c a p to e th a n o l.
G la s s - D i s t i l l e d W a te r
D is tille d w a te r w a s r e d is tille d w ith a C o rn in g M o d el AG-2. W a te r
D is tilla tio n A p p a ratu s (C o rn in g G la s s W o rk s) and. th e d i s t i l l a t e c o lle c te d
. in te n - g a llo n P y rex c a r b o y s . T his w a te r w a s u s e d to p re p a re a ll th e
b u ffe rs u s e d in. th is, in v e s tig a tio n .
Two g ra m s o f B en to n ite. (F is h e r S c ie n tific ) w a s s u s p e n d e d in 40 mi
o f gd w a te r th e n , c e n trifu g e d fo r 15 m in u tes, a t 2500 rpm (800 x g ) in th e
S S -I ro to r o f-th e S e rv a ll c e n tr if u g e . ( J . S o r v a ll, I n c . ) . The s u p e r n a ta n t
fr a c tio n from t h i s s te p w a s c e n tr ifu g e d .fo r 20 m in u te s a t 8500 rpm
(9 2 0 0 x g) in th e S S -I ro to r o f th e S e rv a ll c e n tr ifu g e . The s e d im e n t w a s
r e s u s p e n d e d in gd w a te r fo r 4 8 h o u rs a t room te m p e ra tu re .
The re s u s p e n d e d , b e n to n ite w a s c e n trifu g e d fo r 15 m in u te s a t
2 5 0 0 rp m , fo llo w e d by c e n tr ifu g a tio n o f th e s u p e r n a ta n t fr a c tio n for 20
m in u te s a t 8500 rpm .(b o th in th e S e rv a ll S S -I r o to r ) . The s e d im e n t w a s
a g a in , re s u s p e n d e d , b y s ta n d in g in g d w a te r fo r 48 h o u rs a t room te m p e ra ­
tu r e .
F in a lly , th e r e s u s p e n d e d b e n to n ite w a s c e n trifu g e d fo r 3 0 m in u te s
in th e S S -I ro to r o f th e S e rv a ll; th e s u p e r n a ta n t fra c tio n , d is c a rd e d ; th e
s e d im e n t s u s p e n d e d in gd w a te r; a n d .b o ttle d .
'
G l a s s - D i s t i l l e d H y d ro c h lo ric Acid
R e a g e n t-g ra d e h y d ro c h lo ric a c id (D uPont) w a s d ilu te d I; I w ith gd
w a te r a n d d i s t i l l e d . The f ir s t th ir d .o f th e d i s t i l l a t e w a s s a v e d fo r g e n ­
e r a l la b o ra to ry u s e ; th e m id d le th ird w a s sa v e d , a n d u s e d fo r m aking
buffers, th ro u g h o u t th is in v e s tig a tio n ; a n d .th e la s t- th ir d , d is c a r d e d .
9
R ib e n u c le a s e R em oval
R ib o n u c le a s e (RNAase) w as. rem oved, from g la s s w a r e b y e ith e r:
a) h e a tin g in a d ry o v e n a t 2 4 0 °C fo r 10 h o u rs; o r b) a u to c la v in g , fo r 20
m in u te s . RNAase w a s rem o v ed from, p l a s t i c ware, b y e ith e r: a) a u to ­
c la v in g fo r 20 m in u te s (a u to c la v a b le p l a s t i c o n ly ); o r b) tre a tm e n t w ith
15% h y d ro g e n p e ro x id e fo r 15 m in u te s .
R N A ase:w a s rem o v ed from b u ffe r s o lu tio n s b y m ix in g in 2 d ro p s p e r
l i t e r o f s o lu tio n b e n to n ite a f te r the. b u ffe r w as. p re p a re d . The b u ffer th u s
tr e a te d w a s f ilte r e d th ro u g h , a M illip o re f iltr a tio n a p p a r a tu s (M illip o re
C o rp .) e q u ip p e d w ith a. NAWP 047 M illip o re f i l t e r , a n d c o lle c te d in a
P y rex b o ttle (th e e n tire f iltr a tio n a p p a r a tu s h a d b e e n tr e a te d to rem ove
RNAase p rio r to u s e ) .
. R ibosom e P re p a ra tio n
All r e a g e n t s , g la s s w a r e , c e n trifu g e tu b e s , e t c . to .b e u s e d in th e
p re p a ra tio n w e re m ade s t e r i l e a n d .RNAase fre e by th e a p p ro p ria te t r e a t ­
m e n t ( s e e " R ib o n u c le a se . R e m o v a l" ).
Two- h u n d re d gram s o f fro z e n E s c h e r ic h ia c o li s tr a in Q-13 (G e n e ra l
B io c h e m ic a ls , I n c .) w e re g ro u n d in th re e p o rtio n s in a la rg e m o rtar an d
p e s t l e u n til th e c e l l s w e re s lig h tly th a w e d . A p p ro x im ately 70gm. o f
a lu m in a (N o rto n , C o ., le v ig a te d ) w e re a d d e d to e a c h p o rtio n and. th e
m ix tu re g ro u n d to a th ic k p a s t e . A p p ro x im a te ly 35 ml o f b u ffe r T+ w ere
10
. a d d e d to e a c h portion and. th e m ix tu re g ro u n d to a s lu r r y . T h is p ro c e d u re
y ie ld s a p p ro x im a te ly 35® m l t o t a l v o lu m e .
. The g ro u n d h o m o g e n a te w a s th e n p la c e d in c e n tr ifu g e tu b e s an d
c e n tr ifu g e d 40 m in u te s a t IS5SOGrpm (29,000 x g ) in th e S S -3 4 ro to r o f th e
S o rv a ll RC2-B c e n t r i f u g e d . S o r v a ll, C o .) to rem ove, th e a lu m in a a n d c e ll
d e b r i s . The s u p e r n a ta n t fr a c tio n w a s rem o v ed a n d 0.6 mg o f
d e o x y rib o n u c le a s e I (D N A a se ).(W o rth in g to n B io c h e m ic a ls) w e re a d d e d
a n d s tir r e d into- this, f r a c tio n . The s u p e r n a ta n t fr a c tio n w a s k e p t a t 4 °C
fo r 90 m in u te s to a llo w c o m p le te d ig e stio n , o f th e DNA. The D N A ase
-tre a te d fr a c tio n w a s -then c e n trifu g e d , in th e 30 ro to r o f th e B eckm an
(S p in co D iv is io n ) M o d el L -2 u ltr a c e n trifu g e a t 18,000 rpm (30,0.00x g ) fo r
2, h o u r s . The s u p e r n a ta n t fr a c tio n w as rem o v ed and. tr a n s f e r r e d to tu b e s
s u ita b le fo r th e 5O-Ti r o t o r , th e n c e n trifu g e d fo r 4 h o u rs a t 50,000 rpm
(170,0.00 x g) in th e M o d el L -2 u ltr a c e n t r if u g e . The to p 90% o f th e s u p e r ­
n a ta n t fr a c tio n w a s rem o v ed w ith a s y rin g e a n d s a v e d as. S - 1 0 0 .
F iv e m illilite r s o f b u ffe r OC
w e re a d d e d to e a c h tu b e an d th e
p e l l e t a llo w e d to d is s o lv e o v e rn ig h t w ith o c c a s io n a l stirrin g , o f th e
p e l l e t s u r f a c e w ith a stirrin g , r&d.
T he S -1 0 0 w a s th e n d ia ly s e d 12 -13 h o u rs v e r s u s BC+ b u ffe r a t
4 ° C , b o ttle d , a n d s to re d a t - 9 0 °C u n til u s e d to p re p a re c o n c e n tra te d
S -1 0 0 .
The tu b e s c o n ta in in g th e d is s o lv e d p e l l e t w e re -th e n f ille d w ith
b u ffe r O C + a n d c e n trifu g e d fo r 15 m in u te s a t 2 5 ,0 0 0 rpm (4 2 ,0 0 0 x g ) in
11
th e T ype SO-=Ti ro to r to rem o v e h e a v y m a te r ia l. The s u p e r n a ta n t fra c tio n
w a s th e n la y e r e d on a d ie th y la m in o e th y l. (DEAE)- c e l l u l o s e colum n, p r e ­
v io u s ly p re p a re d , ( s e e " DEAE C olum n P re p a ra tio n " ) and. th e colum n
w a s h e d w ith 2 l i t e r s o f b u ffe r OC .. The rib o s o m e s w e re e lu te d from th e
c o lu m n w ith b u ffe r OD+ an d c o n c e n tr a te d 'b y c e n tr ifu g a tio n a t SOzOOOrpm
. fo r 4 h o u rs in th e SQ-Ti ro to r o f th e M o d el L - 2 .
The. rib o s o m e s w e re d is s o lv e d in a s m a ll am o u n t o f b u ffe r BC,
th e ir c o n c e n tra tio n d e te rm in e d b y a b s o rp tio n , a t 2 60 mU a n d th e ir c o n ­
c e n tr a tio n a d ju s te d to 2 5 o r 50 m g /m l. The rib o s o m e s w e re th e n b o ttle d
. in s m a ll a liq u o ts (2 5 0 -5 0 0 0 yl) a n d k e p t fro z e n a t -9 0 ° C p rio r to u s e .
DEAE C olum n P re p a ra tio n
Two h u n d re d g ram s o f DEAE c e ll u lo s e .(C al-B io ch em ) w ere w a s h e d
w ith 2 l i t e r s o f 0.1 N so d iu m h y d ro x id e b y s u c tio n f i l tr a tio n , w a sh e d to
n e u tr a lity w ith gd w a te r , w a s h e d w ith 2 l i t e r s 95% e th a n o l , an d r e ­
w a s h e d w ith gd w a te r . The r e s in w a s th e n s u s p e n d e d in gd w a te r an d
a u to c la v e d fo r 20 m in u te s .. The r e s in w a s p o u red in to a Type KS-IOO
P h a rm a c ia c o lu m n (5 .0 x 1 0 0 cm) and. p a c k e d dow n u n d e r IO p s i n itro g e n
. p r e s s u r e . The co lu m n w a s th e n W ashed w ith 4 lite r s o f b u ffe r OC+ . The
f in a l d im e n s io n s o f th e r e s in w e re 6 1 x 5 cm .
. S-1.00 C o n c e n tra tio n
F iv e m illilite r s o f b o ttle d S -1 0 0 w e re th a w e d a n d m ad e 76% s a t u ­
r a te d in am m onium s u lf a te (M ann R e s e a rc h C h e m ic a ls , e n zy m e g rad e)
. 12
by s lo w a d d itio n o f th e s o lid . (2.5 g m ). The m ilk y s u s p e n s io n w a s c e n tr i­
fu g ed fo r 20 m in u te s a t 13,500rpm (2 2 ,0 0 0 x g ) in th e SS--34 ro to r o f th e
S o rv a ll R C 2-B . The s u p e r n a ta n t fr a c tio n w a s d is c a r d e d a n d th e p r e c ip i­
ta te d is s o lv e d in b u ffe r BC: th e s o lu tio n w a s th e n d ia ly s e d v e r s u s BC+
: (2 lite r s ) fo r 8-10 h o u r s . A fter d ia l y s i s th e s o lu tio n w a s u s e d im m e­
d ia te ly .
P h e n o l E x trac tio n , o f P o ly c y tid y lic A cid
F ifty m illig ra m s o f p o ly c y tid y lic a c id (polyC ) (M ile s C h e m ic a ls)
w e re d is s o lv e d in 2 ml gd. w a t e r , e x tr a c te d 2, tim e s .w ith w a te r s a tu r a te d
p h e n o l (M a llin c k ro d t gold, l a b e l ) , an d e x tr a c te d 3 tim e s w ith d ie th y l
e th e r . The aq u eo u s, la y e r w a s th e n m ad e 0.5 M .in .p o ta s s iu m c h lo rid e b y
a d d itio n o f th e s o l i d . The. p o ly C w a s p r e c ip ita te d by m ak in g th e s o lu ­
tio n 60% (v /v ) in e th a n o l. The p r e c ip ita te w a s w a s h e d s u c c e s s iv e ly
w ith 80%. e t h a n o l , 100% e t h a n o l , an d d ie th y l e tiie r b y s tir r in g th e p r e ­
c ip ita te w ith th e w a s h in g a g e n t a n d s e d im e n tin g th e p re c ip ita te , in a
c li n i c a l c e n trifu g e (I n te r n a tio n a l E q u ip m en t C o .,^M o d el C L ). The r e ­
s u ltin g .p re c ip ita te w a s th e n d rie d o v e rn ig h t in a v acu u m d e s s i c a t o r
c o n ta in in g c a lc iu m c h lo r id e .
In c o rp o ra tio n P ro c e d u re
The. in c o rp o ra tio n o f ^ C - p r o l i n e in to p e p tid e s w a s c a rrie d o u t b y
. in c u b a tin g th e fo llo w in g m a te ria ls in 12 ml c o n ic a l c e n tr ifu g e tu b e s a t
- 3 7 °C : 1.0 uM ATP; 0.03 uM GTP; 1.0 uM ’.PEP; 0.3 E .U .,(lug) P y ru v ate
13
k in a s e ; 0.1 nC i ^ C - p r o l i n e (255 m C i/m M ) A m ersh am = S earle C o rp .);
100 Ml c o n c e n tr a te d S -1 0 0 ; 0.4 mg E . c o ll t-RN A (S ch w a rz B io r e s e a r c h ,
In c .); a p p ro x im a te ly 1.25 mg rib o s o m e s ; 6 Hg o f p h e n o l e x tr a c te d p o ly C .
The ATP, GTP, PEP, a n d p y ru v a te k in a s e w e re o b ta in e d from C a l B io c h em .
All th e a b o v e m a te r ia ls w e re c o n ta in e d in I ml o f b u ffe r B C .
The in c o rp o ra tio n w a s p erfo rm ed b y a d d in g to e a c h tu b e a ll th e
a b o v e m a te r ia ls from s to c k s o l u t i o n s , l e s s p o ly C . C h lo ra m p h e n ic o l
(d o n a te d by P a rk e -D a v is C o.) w a s a d d e d in th e c o n c e n tr a tio n s in d ic a te d .
In c o rp o ra tio n w a s i n itia te d by a d d itio n o f p o ly C a t tim e d in te r v a ls an d
in c o rp o ra tio n w a s a llo w e d to p ro c e e d fo r 40 m in u te s .. The in c o rp o ra tio n
w a s s to p p e d by th e a d d itio n o f 6 ml o f 20% tr ic h lo r o a c e tic a c id (TCA).
P la tin g P ro c e d u re fo r th e
14
C -P ro lin e P e p tid e s
The tu b e s from th e in c o rp o ra tio n w e re a llo w e d .to s ta n d fo r a t l e a s t
30 m in u te s a t 4 °C a f te r a d d itio n o f 20% TCA; The p r e c i p i t a t e w a s th e n
c e n trifu g e d for 5 m in u te s a t top s p e e d in a c lin ic a l c e n tr ifu g e and. th e
s u p e r n a ta n t liq u id re m o v e d by a p a s te u r p ip e tte a tta c h e d to a n a s p ir a to r .
The p r e c ip ita te w a s th e n d is s o lv e d in 2 ml 0.2 M p o ta s s iu m h y d ro x id e a n d
r e p r e c ip ita te d w ith 6 ml 20% TCA. The p r e c ip ita te w a s a llo w e d to f lo c ­
c u la te for a t l e a s t 30 m in u te s , th e n c o lle c te d on M illip o re filte rs, (HAWP
024), w a s h e d w ith. 20% TCA, re m o v e d , a n d g lu e d to c o p p e r d is k s , p la c e d
in d is c h o ld e rs (N u c le a r N ic a g o C o rp .), d r i e d , an d c o u n te d in a N u c le a r
C h ic a g o lo w b a c k g ro u n d p la n c h e tte c o u n te r .
14
P a rtia l, A cid H y d ro ly z a te o f Po ly p ro lin e
IiiiMIIIIrtvIii I ir p iX M C T f a o ta n c ^ ^ M O im ^ m g M ^ -
T w en ty m illig ra m s o f L -p o ly p ro lin e (M ile s C h e m ic a ls ) w a s d i s ­
s o lv e d in 4 m l o f gd H C l, p la c e d in a th ic k - w a lle d c e n tr ifu g e tu b e f itte d
w ith a v a cu u m a d a p to r , a n d e v a c u a t e d „ The s o lu tio n w a s h e a te d a t 9 0 °C
fo r 90 m in u te s in a h e a tin g b lo c k . The s o lu tio n w a s c o o l e d , ly o p h iliz e d ,
a n d d is s o lv e d in I ml o f gd w a t e r .
P re p a ra tio n o f th e
14
C -P ro lin e P e p tid e s for A n a ly s is
The in c o rp o ra tio n w a s s to p p e d b y c h illin g th e tu b e s i n a n ,ic e b a th .
E x tra n e o u s p ro te in s w e re re m o v e d b y h e a tin g th e tu b e s in a m ild ly b o il­
in g w a te r b a th fo r 2 m in u te s fo llo w e d b y c h illin g in a n ic e b a th . The
p r e c ip ita te w a s th e n rem o v ed by f iltr a tio n o n M illip o re f il te r s (HAWP
0 2 4 ). The s a m p le s w e re th e n ly o p h iliz e d . to d ry n e s s , d is s o lv e d in 0.5 ml
0.2 N- p o ta s s iu m h y d ro x id e , 800 Hg o f p o ly p ro lin e p a r tia l a c id h y d ro ly z a te
a d d e d , a n d th e s a m p le s r e ly o p h iliz e d to d r y n e s s . The ly o p h iliz e d
m a te ria l w a s th e n , s u s p e n d e d in 0.1 ml o f 1% T rito n X -1 0 0 (A lk y lp h en o x y
. p o ly e th p x y e th a n o l, Rohm an d H a a s C o.) fo r s p o ttin g .
C o m b in ed E le c tro p h o re s is -C h ro m a to g ra p h y o f th e P ro lin e P e p tid e s
The sa m p le w a s s p o tte d 3 cm from tw o e d g e s o f "Whatman 3MM
p a p e r s h e e t ( 4 0 x 2 5 cm) w ith a 5 p i m ic ro p ip e tte . The s h e e t w a s th e n
w e tte d w ith th e b u ffe r s o lu tio n o f 4% fo rm ic a c id a n d b lo tte d th o ro u g h ly .
The s h e e t w a s th e n p la c e d on th e p la te o f a h ig h - v o lta g e e le c tro p h o r­
e s is . a p p a r a tu s . (M o n ta n a S ta te U n iv e rs ity In s tru m e n t S e rv ic e ) an d
15
c o n n e c te d to th e b u ffe r c h a m b e r s , c o n ta in in g a p p ro x im a te ly 900 ml o f 4%
form ic a c id e a c h , b y w ic k s o f W h atm an 3M M p a p e r w ith c e ll u lo s e R is k ­
in g a s m em brane d iv id e r b e tw e e n w ic k s a n d p a p e r . The c o o lin g w a te r
w a s tu rn e d on an d 4 p s i a p p lie d to th e p a p e r b y th e to p c o o lin g b ag o f
th e e le c tr o p h o r e s is a p p a r a t u s . The e n tir e s y s te m w a s a llo w e d to e q u ili­
b ra te 15-30 m in u te s . A fter e q u ilib ra tio n . 1500. v o lts w e re a p p lie d a c r o s s
the. jaaper fo r 3^2 h o u rs . U pon c o m p le tio n o f .e le c tro p h o r e s is , th e p a p e r
w a s rem o v ed a n d d ried, b y h a n g in g in a fum e h o o d .
The d rie d p a p e r w a s r o lle d in to a c y lin d e r,, s t a p l e d , a n d p la c e d in
a c h ro m a to g ra p h y ta n k c o n ta in in g fr e s h b u ta n o l: a c e t i c - a c i d : w a te r
(5 :1 :4 , v / v / v ) t o p . la y e r . The c h ro m ato g ram w a s th e n d e v e lo p e d in a n
a s c e n d in g m a n n er fo r 12 h o u rs . A fte r.th is tim e th e p a p e r w a s re m o v e d ,
d rie d in a fum e h o o d , a n d th e c a r r ie r p e p tid e s d e te c te d b y sp ra y in g w ith
1% n in h y d rin (E astm a n Kodak C o.) in g la c i a l a c e tic a c id fo llo w e d by
h e a tin g a t .6 0 -5 0 °C fo r 3 -5 m in u te s .
R a d io a u to g ra p h P ro c e d u re '
The p a p e r from th e c o m p le te d e le c tro p h o r e s is -c h ro m a to g ra p h y s te p
w a s c u t in to tw o e q u a l s h e e ts 2 0 x 2 0 cm a n d p l a c e d in. film h o ld e rs
(G e n e ra l E le c tric C o ., E a stm a n Kodak C o ., an d hom e m ad e) w ith a s h e e t
o f X - r a y film. (E astm a n K odak, no s c re e n ) a n d a llo w e d to d e v e lo p fo r 7
d a y s . The film w a s d e v e lo p e d fo r 5 m in u te s in ra p id X -ra y d e v e lo p e r
. (G e n e ra l E le c tric C o .) , s to p p e d in d ilu te a c e tic a c id , and. fix e d fo r 5
16
m inutes- in-,rapid X -ray f ix e r (G e n e ra l E le c tric C o .) . The film was- w a s h e d
in ta p w a te r fo llo w e d b y a d i s t i l l e d w a te r w a s h an d d r ie d .
/
,
. Q u a n tita tiv e D e te rm in a tio n o f th e S e p a ra te d ^ C - P r o l i n e P e p tid e s
The. a r e a s o f th e c o m b in e d e le c tro p h o r e s is -c h ro m a to g ra m ,(map) i n ­
d ic a te d to c o n ta in p ro lin e p e p tid e s a s d e te rm in e d e ith e r from th e ra d io a u to g ra m dr. from th e n in h y d rin r e a c tio n w ith c a r r ie r p e p tid e s w e re c u t o u t
a n d p la c e d in s c i n t illa tio n v i a l s (B e c k m a n ). F ifte e n m illilite r s o f s c in ­
t i lla tio n flu id c o n ta in in g 4g m 2 ,5 - d ip h e n y lo x a z o le a n d 5 0 mg. l , 4 - b i s ^ 2 . ( 5 - p h e n y lo x a z o ly l) - b e n z e n e p e r l i t e r o f to lu e n e m ade b y d ilu tio n o f
L iq u iflu o r (N u c le a r C h ic a g o C o r p .) 42 m l / l i t e r w a s added, to e a c h v i a l .
The s a m p le s w e re c o u n te d in a N u c le a r C h ic a g o liq u id s c in t illa tio n
s p e c tro m e te r,(S y s te m 6804) fo r 10. m in u te s .
S u c ro se ' D e n s ity G ra d ie n t A n a ly sis
The s a m p le s fo r a n a l y s i s w e re o f tw o ty p e s : a) 250 p.1 o f a s t a n 3
d a rd in c o rp o ra tio n m ix tu re; b) 0.6 pC i o f H -p o ly C (11.81 m C i/m M p h o s ­
p h a te ) (M ile s L a b o rato ries.) in c u b a te d w ith 1.25 mg, o f rib o s o m e s for 5
m in u te s a t .370 C in a to ta l volum e, o f 200 |Ul b u ffe r B C .
. The s a m p le s w e re la y e r e d o n th e to p o f a .5-20% s u c r o s e g ra d ie n t
p re p a red , b y ru n n in g 16 ml o f a 2 0 -5 % lin e a r s u c r o s e g ra d ie n t in to c e ll u ­
lo s e n itr a te tu b e s (B eckm an C o rp .) w h ic h c o n ta in e d a I ml c u s h io n o f
50% s u c r o s e . The g r a d ie n ts w e re c e n trifu g e d a t 2 5 ,0 0 0 rpm (7 5 ,0 0 0 x g )
fo r 4V2 h o u rs in th e B eckm an SW 2 5.3 ro to r .(S p in co -D iv isio n ) o f th e
17
M o d el L -2 u ltr a c e n t r if u g e . The g ra d ie n ts w e re f r a c tio n a te d in to 16 -d ro p
fr a c tio n s in to s c in tilla tio n , v i a l s b y m e a n s o f a n LKB d ro p c o u n te r (LKB~
P ro d u k te r, R adiR ac T ype 342 BA) and. LKB. fra c tio n c o lle c to r (LKBP ro d u K ter, R adiR ac Type 3401A) from th e b o tto m o f e a c h tu b e , a 100-ul
sa m p le w a s re m o v e d from e a c h ,v ial a n d .d ilu te d to I ml w ith gd w a te r.
The re m a in d e r o f e a c h , fr a c tio n w a s d is s o lv e d in 15 ml o f s c in t illa tio n
fluid. (1100 ml to lu e n e ; 900 ml e th a n o l; 168 ml form am ide; 6.4 gm 2 ,5 d ip h e n y lo x a z o le ) a n d c o u n te d in a N u c le a r C h ic a g o liq u id s c in t illa tio n
s p e c tro m e te r for 10 m in u te s . The o p tic a l d e n s ity o f th e d ilu te d fra c tio n s
w a s d eterm in ed , a t 2 60 mu in a G ilfo rd m u ltip le - s a m p le a b s o rb a n c e r e ­
c o rd e r e q u ip p e d w ith a B eckm an D U -R m o n o c h ro m e te r.
CHAPTER III
■ EXPERIMENTAL RESULTS
S ta n d a rd p ro lin e p e p tid e s ( C y c lo C h e m ic a l C o.) w e re s e p a r a te d b y
h ig h - v o lta g e e le c tr o p h o r e s is in 4% fo rm ic a c id . (F ig . I) b u t p ro lin e
.(N u tritio n a l B io c h e m ic a ls C o.) w a s .n o t re s o lv e d fro m .th e tr ip e p tid e .
C h ro m a to g ra p h y y ie ld e d c o m p le te s e p a ra tio n , o f p ro lin e from i t s p e p tid e s
(F ig . 2 ). The c o m b in a tio n o f th e s e tw o te c h n iq u e s y ie ld e d c o m p le te s e p ­
a r a tio n o f th e p e p tid e s s m a lle r than, th e h e p ta p e p tid e a s sh o w n in F ig . 3 .
The c e ll- f r e e , in c o rp o ra tio n s y s te m w a s c h a r a c te r iz e d a s to th e
e f f e c t o f m agnesium , io n c o n c e n tra tio n ( F ig .. 4) an d p o ly C c o n c e n tra tio n
( F ig . 5 ). The d is trib u tio n , o f th e ^ C - p r o l i n e p e p tid e s s y n th e s iz e d in
v itr o a s d ire c te d , by p o ly C w a s d e te rm in e d b y a u to ra d io g ra p h y o f a p e p ­
tid e m ap a s d e p ic te d in F ig . 6. Two unknow n, ra d io a c tiv e s p o ts a p p e a re d
in th e a u to ra d io g ra m w h ich ; d id n o t c o rre s p o n d to -th e n in h y d rin p o s itiv e
c a r r ie r p ro lin e p e p t i d e s .. The un k now n s p o t a t .the. o rig in o f th e map w a s
. sh o w n to c o rre s p o n d to the. 15% TCA p r e c ip ita b le p e p tid e s (F ig . 7).
• P a r tia l a c id h y d r o ly s is o f th e 15% TCA p re c ip ita b le p e p tid e s y ie ld s a d i s ­
trib u tio n o f p e p tid e s w h ic h c o rre sp o n d , to. a d d e d c a r r ie r p ro lin e p e p tid e s
a s sh o w n in .F ig . 8 . Thus th e p e p tid e s rem aining, a t th e o rig in w ere d e s ­
ig n a te d p o ly p ro lin e (P P ). The o th e r u n k n o w n s p o t w h ic h re m a in e d a t th e
i •
o rig in d u rin g e le c tr o p h o r e s is b u t m ig ra te d n e a r th e s o lv e n t .front .upon
. c h ro m a to g ra p h y w a s is o la te d from a map a n d w a s sh o w n to m ig ra te th e
sa m e d is ta n c e a s a s ta n d a rd o f L -p ro ly lp ro lin e d ik e to p ip e ra z in e . o n a
19
c e ll u lo s e th i n - l a y e r s h e e t (B rin k m an , MN c e l .300) in b u ta n o l: a c e tic
a c i d : w a te r ( 5 :1 :4 , v / v / v ) to p p h a s e . A lso m ix in g th e u n k now n w ith
s ta n d a rd DKP fo llo w e d by th i n - l a y e r c h ro m a to g ra p h y on a s i l i c a g e ll
s h e e t (B rinkm an, SlL S-HR) in c h lo ro fo rm : m ethanol. ( 9 : l , . v / v ) , sh o w ed
t h a t th e ra d io a c tiv e unknow n, m ig ra te d to e x a c tly th e sa m e p o s itio n a s
th e s ta n d a rd DKP, The s ta n d a rd DKP w a s d e te c te d b y s p ra y in g w ith 1%
.t-b u ty lh y p o c h lo rite , (F rinton. C h e m ic a ls ) in l,2 -d ic h lo r6 e th a fie -.l0 % a c e t i c
a c id fo llo w e d by s p ra y in g w ith 1% s ta rc h -1 % p o ta s s iu m io d id e (4 9 ). The
r a d io a c tiv e unknow n w a s d e te c te d b y a u to ra d io g ra p h y .
S u c ro s e d e n s ity g r a d ie n t a n a ly s is o f a s ta n d a rd in c o rp o ra tio n r e ­
v e a ls th a t- th e p o ly C -p ro lin e s y s te m is a n in v itro p o ly so m e s y ste m (F ig .
3
9) T his o b s e r v a tio n w a s c o n firm e d u p o n a n a ly s is o f th e H -p o ly C rib o so m e c o m p le x b y s u c r o s e d e n s ity g r a d ie n t.a n a ly s is a s sh o w n in
F ig . 1 0.
The. in h ib ito r y e f f e c t o f CAP on th e s y n th e s is o f th e 20% TCA p r e c ip ita b le p e p tid e s is show n:.in F ig . 11.
The e f f e c t o f CAP on. th e fo rm atio n o f a ll the; p e p tid e s is show n fo r
fo u r d iff e r e n t c o n c e n tr a tio n s o f CAP in F i g s . 12 ,1 3 , 14., 15 . In th e s e , fig ­
u re s -th e f r a c tio n s 2 to 6 c o rre s p o n d to s e p a r a te d p e p t i d e s , d ip e p tid e to
h e x a p e p tid e r e s p e c t i v e l y . The fra c tio n n u m b ers 7 to 14 c o rre sp o n d o n ly
to s u c c e s s i v e 2 cm f r a c tio n s rem o v ed from, th e m aps from b e h in d th e r e ­
s o lv e d h e x a p e p tid e . The a b n o rm a lly h ig h v a lu e s o f th e p e p tid e s p ro ­
d u c e d in th e p r e s e n c e o f CAP in th e fr a c tio n 8 an d 9 o f F ig . 14 a re
20
a ttr ib u te d to a c o n ta m in a tio n o f so m e s o r t in th is re g io n o f th e m ap .
T h is i s a p p a r e n t from th e s h a p e o f th e r a d io a c tiv ity in th a t a re a o f th e
m ap a s ,v is u a liz e d b y th e ra d io a u to g ra m o f th e m a p ,
. F in a lly , in F ig . 1 6 , a c o m p a ra tiv e p lo t is p r e s e n te d sh o w in g
CAP'S in h ib itio n o f th e fo rm a tio n .o f p o ly p ro lin e , to ta l p e p t i d e s , a n d 20%
TCA p r e c ip ita tb le p e p t i d e s .
21
1
2
3
4
5
F ig u re I .
E le c tro p h o re s is o f P ro lin e a n d P ro lin e P e p tid e s .
I) P r o lin e , 2) d ip r o lin e , 3) p a r tia l a c id h y d ro ly s a te o f p o ly p ro lin e ,
4) trip ro lin e , 5) te t r a p r o l i n e .
22
F ig u re 2 .
P a p e r C h ro m a to g ra p h y o f P ro lin e an d P ro lin e P e p t i d e s .
D e v e lo p e d in b u ta n o l: a c e t i c a c i d : w a te r , 5 :1 :4 , u p p e r p h a s e I) t e t r a p r o li n e , 2) trip ro lin e , 3) d ip ro lin e , 4) p r o li n e .
c h ro m a to g ra p h y
23
e le c tr o p h o r e s is
F ig u re 3 .
C o m b in ed E le c tro p h o re s is - C h r o m a to g ra p h y o f a P a rtia l A cid
H y d ro ly s a te o f P o ly p r o lin e .
24
M a g n e siu m io n c o n c e n tra tio n (mM)
F ig u re 4 .
tio n o f
14
The E ffe c t o f M a g n e siu m Io n C o n c e n tra tio n o n th e In c o rp o ra C -P ro lin e in to P r e c ip ita b le P e p t i d e s .
25
P o ly c y tid y lic a c id (n g /m l)
F ig u re 5 .
p o ra tio n o f
The E ffe c t o f P o ly c y tid y lic A cid C o n c e n tra tio n o n th e In c o r14
C -P ro lin e in to P r e c ip ita b le P e p tid e s .
c h ro m a to g ra p h y
26
e le c tr o p h o r e s is
F igure 6 .
A u to rad io g ram o f th e 14C -P r o lin e P e p tid e s S y n th e s iz e d in
v itr o D ire c te d by P o ly C .
S e p a ra tio n b y c o m b in e d e le c tr o p h o r e s is - c h r o m a to g r a p h y a s in F ig . 3 .
c h ro m a to g ra p h y
27
e le c tr o p h o r e s is
F ig u re 7 .
A u to rad io g ram o f th e 15% TCA P r e c ip ita b le P e p tid e A n aly sed
by C o m b in ed E le c tr o p h o r e s is - C h r o m a to g r a p h y .
c hrom a to g ra p hy
28
e le c tr o p h o r e s is
F ig u re 8 .
A u to rad io g ram o f th e 15% TCA P r e c ip ita te S u b je c te d to P a rtia l
A cid H y d ro ly s is a s A n a ly se d by C o m b in ed E le c tr o p h o r e s is C h ro m a to g ra p h y .
c o u n ts /m in u te x 10
29
fr a c tio n n u m b er
F ig u re 9 .
S u c ro s e D e n s ity G ra d ie n t A n a ly s is o f a S ta n d a rd In c o rp o ra ­
tio n (250 |0l).
30
10
0
1
O
8
4
I
c o u n ts /m in u te x 10
o p tic a l d e n s ity (260 mu)
•
CO
0
fr a c tio n n u m b er
F ig u re 10.
S u c ro s e D e n s ity G ra d ie n t A n a ly s is of th e
C o m p lex In c u b a te d w ith P u rifie d R ib o s o m e s .
3
H -P o ly C -R ib o so m e
In h ib itio n
31
C h lo ra m p h e n ic o l (u g /m l)
F ig u re 11.
The E ffe c t o f C h lo ra m p h e n ic o l on th e In c o rp o ra tio n o f 14C -
P ro lin e in to P re c ip ita b le P e p tid e s .
32
CO
12
-
8
-
c o u n ts /m in u te x 10
'o
3
C
!
4 -1
C
D
O
U
4 -
2
3
4
.[| M i k l i M - I -1 1 1
5
6
7
8
9
10
11 12 13
14
PPDKP
P e p tid e F ra c tio n
F igure 12 .
The E ffe ct o f C h lo ra m p h e n ic o l on th e D is trib u tio n o f
P ro lin e P e p tid e s ( I p g / m l ) .
I
I c o n tro l
g g H
cap
C-
33
12
CO
c o u n ts /m in u te x 10
I
X
CD
+ ->
3
C 8
E
4co
-J
C
3
O
U
4
0
2
3
4
5
6 * 7
8
9
10 11
12 13
14
P e p tid e F ra c tio n
F igure 13.
The E ffe c t o f C h lo ra m p h e n ic o l on th e D is trib u tio n o f
P ro lin e P e p tid e s (5 p g /m l) .
I
___I c o n tro l
CAP
~C-
34
12
-
8
-
CO
3
C
-H
c o u n ts /m in u te x 10
'o
M
c
3
O
O
4-
O
6*7
8
9
10
11
12
13
PP DKP
P e p tid e F ra c tio n
F ig u re 14.
The E ffe c t o f C h lo ra m p h e n ic o l on th e D is trib u tio n o f
P ro lin e P e p tid e s (10 p g /m l) .
I
I c o n tro l
i H H H CAP
C-
35
-4 0
-3 0
12
I
i _
x 10
CO
CO
(U
+J
-20
c 8
c
E
G
3
O
O
C
D
O
O
4
UML
0 -u -
PP DKP
F igure 15.
The E ffe c t o f C h lo ra m p h e n ic o l on th e D is trib u tio n o f ^ C -
P ro lin e P e p tid e s (25 |u g /m l).
3 c o n tro l
’'
CAP
36
20 —
C h lo ra m p h e n ic o l ( |ig /m l)
F ig u re 16.
0
The E ffe c t o f C h lo ra m p h e n ic o l on P ro lin e P e p tid e S y n th e s is .
p o ly p ro lin e — from p e p tid e map
Ak to ta l p e p tid e s — from p e p tid e map
O
15% TCA p r e c ip ita te — d u p lic a te s a m p le s
C H A P T E R IV
DISCUSSION
■ The d is tr ib u tio n o f th e m a jo r p ro d u c ts o f th e ^ C ” P ro lin e p e p tid e s
s y n th e s iz e d .i n . v itro a s d ir e c te d b y p o iy C a s sh o w n in F ig , 12,is n o t c o n ­
tin u o u s . The m a jo r p ro d u c ts o f th e s y s te m a re th e DKP, th e d i p e p tid e ,
th e t r i p e p t i d e , a n d p o ly p ro lin e (P P ). P e p tid e s o f in te rm e d ia te s iz e b e ­
tw e e n th e s m a ll p e p tid e s (DKP, d i p r o l in e , trip ro lin e ) an d th e la rg e
p e p tid e s , (p o ly p ro lin e ) a re form ed in r e la tiv e ly s m a ll a m o u n ts in p ro p o r­
tio n to th e o th e r s . T h e re fo re in th e c o u rs e , o f th is d is c u s s io n th e s y s te m
a n d CAP'S e f f e c t u p o n .it w ill b e d is c u s s e d in te rm s o f th e m ajo r p ro d u c ts
th u s d e s ig n a te d .
The o v e r a ll s in g le m a jo r p ro d u c t o f th e s y s te m i s th e c y c lic d ip e p ­
tid e , th e d ik e to p ip e ra z in e . o f L -p ro ly lp ro lin e (DKP). Its fo rm atio n m ay
o c c u r in tw o w a y s: (I) fo rm a tio n u p o n c le a v a g e o f th e a c y l bond o f th e
d ip ro ly l-t-R N A d u rin g th e sa m p le p re p a ra tio n , fo r m ap p in g ; (2) fo rm atio n
in s itu on. th e .rib o s o m e during, p e p tid e s y n t h e s i s . If th e f i r s t c a s e is
tru e then, th e DKP m ay b e c o n s id e re d a s p a rt o f.th e d ip e p tid e p r o d u c t. If
th e s e c o n d c a s e i s .true th e n .the DKP fo rm a tio n m ay h a v e a s ig n if ic a n t
b e a rin g on th e m echanism , o f p e p tid e b o n d fo rm atio n a n d th e e f f e c t o f
CAP upon i t .
. I t i s n o t s u r p r is in g th a t th e DKP i s formed, in t h i s s y s te m s in c e
c y c lic d ip e p tid e s o f th is ty p e a re know n to form .re a d ily from p e p tid e s
c o n ta in in g p ro lin e a n d g l y c i n e , e s p e c i a l l y th o s e c o n ta in in g p r a l i n e .
38
A nother im p o rta n t fe a tu re o f th is s y s te m i s t h a t p o ly C forms, p o ly ­
s o m e s w ith rib o s o m e s in th e p r e s e n c e a n d a b s e n c e o f s u p e r n a ta n t
f a c to r s (F ig s . 9 and. 10). T his f a c t , in i t s e l f , i s a r e le v a n t in d ic a tio n
t h a t th e p o ly C sy ste m , is s im ila r in som e, r e s p e c ts to in v iv o p ro te in s y n ­
t h e s i s s in c e in v iv o p ro te in s y n th e s is h a s b e e n sh o w n to o c c u r a t
p o ly so m e s. (50, 51).
O f th e p o s s ib le e x p la n a tio n s fo r th e o b se rv e d , p e p tid e d is trib u tio n
tw o a re p r e s e n te d a s fo llo w s:
1.
The s m a ll p e p tid e s a re s y n th e s iz e d by a d if f e r e n t m e ch a n ism
th a n a re th e la rg e p e p tid e s . Such a m e c h a n ism w o u ld b e e x p e c te d to b e
a r tif i c ia l in c o m p ariso n , t o n a tu ra l p ro te in , s y n th e s is . The,, la rg e p e p tid e s
w ould, th e n be s y n th e s iz e d by a m e c h a n is m id e n tic a l o r v e ry s im ila r to
in v iv o p ro te in s y n t h e s i s .
2.
L arge a n d s m a ll p e p tid e s a re . form ed by th e sa m e m e ch a n ism
b u t.th e in v itro s y s te m m ay h a v e a te n d e n c y to a b o rt v e ry e a r ly in th e
s y n th e s is p r o c e s s s u c h th a t la rg e , q u a n titie s o f s m a ll p e p tid e s a re
fo rm e d . Thus in. o n ly a m in o rity o f th e in itia tio n s , th e s y s te m is a llo w e d
.to p ro c e ed , to c o m p le te -the. la rg e p e p tid e s .
Both o f- th e s e p o s tu la te s w o u ld se e m to in v o lv e o c c u r re n c e s w h ic h
ta k e p la c e , e a r ly in p ro te in s y n th e s is b u t a f te r fo rm atio n , o f th e in itia tio n
c o m p le x . In t h i s d is c u s s io n th e term in itia tio n , c o m p le x re f e r s to th e
r io b o s o m e - m e s s e n g e r c o m p le x w h ic h in c lu d e s a l l.th e n e c e s s a r y fa c to rs
a n d a m in o a c y l-t-R N A s. p ro p e rly c o m b in e d .to b e g in p e p tid e s y n t h e s i s .
39
The. fo rm a tio n o f th e f i r s t p e p tid e b o n d in a n in v itr o s y s te m o f th is
ty p e i s n o t a n a lo g o u s to th a t o c c u rrin g in b a c te r ia l in v iv o o r in ,vitro
, p ro te in , s y n th e s is d ir e c te d b y n a tu ra l m e s s e n g e r s . In. th e b a c te r ia l s y s ­
tem . n a tu ra l in itia tio n o f.th e f i r s t p e p tid e bond in v o lv e s a n N - a c y la te d
.. a m in o a c y l-t-R N A a s. th e f ir s t am ino a c id in c o rp o ra te d in to th e p e p tid e
..(5 2 ,5 3 ,5 4 ,5 5 ,5 6 ,5 7 ,5 8 ). N a tu ra l in itia tio n i s p e rm itte d a t m uch lo w e r
m a g n e siu m io n c o n c e n tr a tio n s ( le s s th a n 10 mM) th a n re q u ire d fo r i n i t i ­
a tio n w ith s y n th e tic m e s s e n g e r s u s in g u n s u b s titu te d a m in o a c y l-t-R N A .
S in c e -th e . in itia tio n , o f p e p tid e bo n d fo rm a tio n fo r th e p o ly C - s tim u la te d
s y s te m i s n o t " n a tu ra l" o n e m ig h t e x p e c t th a t th e fo rm a tio n o f th e f ir s t
p e p tid e b o n d m ay o c c u r in a d iff e r e n t m a n n er th an , o c c u r s in v iv o . ,If
s u c h i s th e c a s e th e n th e fo rm atio n o f e x c e s s iv e a m o u n ts o f d ip e p tid e
m ay o c c u r b y a m e c h a n ism w h ic h w ill n o t a llo w th e fo rm atio n , o f la rg e
p e p tid e s . L arge p e p tid e s m ay be form ed only, if d ip ro ly l-t-R N A fin d s i t s
w ay to th e d o n o r (peptid.yl) s ite a n d a llo w s in itia tio n , to o c c u r in a " m ore
n a tu ra l" m a n n e r. Y et th e re i s n o 'e v id e n c e : to in d ic a te , t h a t tw o d iffe re n t
m e ch a n ism s, for p e p tid e bond fo rm atio n e x i s t , b u t th e re is no d o u b t th a t
th e re a re o b v io u s d if f e r e n c e s in th e fo rm a tio n o f th e f i r s t p e p tid e bond
in th e s y n th e tic m e s s e n g e r s y s te m c o m p ared to " n a tu ra l" in itia tio n .
The s y n th e s is o f p re c ip ita b le . p e p tid e s o f p ro lin e form ed in th e
p o ly C - s tim u la te d s y s te m i s v e ry s e n s i t i v e to CAP as. sh o w n in F ig . 11.
I n .f a c t , th is in h ib itio n i s lik e th a t ,o b se rv e d fo r-th e in v itr o s y n th e s is o f
M S-2 c o a t p ro te in (27) a n d th e c o n c e n tr a tio n s u s e d a re in th e sam e
40
c o n c e n tra tio n ra n g e a s -th o se re q u ire d to in h ib it th e g ro w th o f b a c te r ia
(5 9 ). T h erefo re th is in h ib itio n o b s e r v e d a t th e sam e c o n c e n tr a tio n s a t
w h ic h CAP in h ib its , i n .y iv o - p ro te in s y n th e s is , is. te rm e d lo w c o n c e n tra tio n
'
_5
in h ib itio n (10
—10
M ). A n a ly s is o f th e to ta l p e p tid e s , p ro d u c e d in .th e
p r e s e n c e o f CAP' re v e a ls , th a t th e fo rm a tio n o f th e la rg e p e p tid e s i s m ore
s e n s i t i v e to i t s a c tio n .th a n i s th e form ation, o f th e s m a ll p e p tid e s . Y et
th e fo rm a tio n o f th e s m a ll p e p tid e s i s s e n s itiv e , to lo w c o n c e n tra tio n s o f
CAP. T his i s in c o n tr a s t to -the o b s e r v e d e f f e c t o f CAP o n th e polyA d ir e c te d .s y n th e s is , of ^ C - I y s i n e p e p tid e s . In th a t s y s te m th e fo rm atio n
o f s m a ll p e p tid e s is n o t in h ib ite d b y h ig h c o n c e n tra tio n s o f CAP a n d th e ir
fo rm atio n i s e v e n s tim u la te d in th e p r e s e n c e o f th e a n tib io tic (2 5 ). A lso
in h ib itio n .o f p ro lin e p e p tid e s y n th e s is , b y a l l c o n c e n tra tio n s o f CAP
s tu d ie d d id n o t r e s u l t in an. a p p r e c ia b le s h iftin g o f th e p e p tid e d is tr ib u ­
tio n . T h e se r e s u lts in d ic a te th a t CAP a c t s a t som e e a r ly step, in th e
fo rm a tio n o f th e p ro lih e p e p tid e s .
S tu d ie s a t h ig h c o n c e n tra tio n s o f CAP (100 u g /m l) h a v e le d m any
a u th o rs to s t a t e t h a t CAP in h ib its p e p tid y l t r a n s f e r a s e , th e en zy m e w h ic h
c a ta ly z e s th e fo rm a tio n o f th e p ep tid e, b o n d (2 9 ,3 1 ,3 2 ,3 3 ). T h e se s tu d ie s
w e re b a s e d on CAP'S in h ib itio n o f th e p u ro m y cin r e a c tio n . Y et o th e r
a u th o rs a rriv e a t an. o p p o s ite c o n c lu s io n from s tu d ie s o n th e sam e ty p e
o f s y s te m ..(34). The l a t t e r a u th o rs s ta te , th a t CAP in h ib its som e s te p
p rio r to th e a c tio n o f p e p tid y l t r a n s f e r a s e . Some, p o s s i b i l i t i e s w h ic h
e x i s t p rio r to th is s te p a re :
41
1.
T r a n s lo c a tio n o f p e p tid y l-t-R N A . from, th e a c c e p to r (a m in o a cy l)
s i t e to th e d o n o r s i t e ,(a fu n c tio n o f th e tr a n s lo c a s e o r G f a c to r ) .
2.
R ib o so m al m o v em en t along, th e m e s s e n g e r .
3.
E x p u lsio n o f d e a c y la te d t-R N A a f te r p e p tid e bond, fo rm a tio n .
4.
B inding o f the. a m in o a c y l e n d o f a m in o a c y l- o r p e p tid y l-t-R N A
to th e 50S s u b u n it o f th e rib o s o m e .
5.
Some a s y e t u n k n o w n .ste p in p ro te in s y n t h e s i s .
A ll-th e a b o v e p o s s i b i l i t i e s in v o lv e so m e fu n ctio n , o f th e SOS s u b u n it on
w h ich . CAP i s sh o w n to a c t .
The m ain d if f ic u lty w ith d e te rm in in g o r p o s tu la tin g a m ech an ism , o f
a c tio n , fo r CAP i s .the f a c t th a t th e m a jo rity o f th e lite r a tu r e re p o rte d is
b a s e d o n .o b s e r v a tio n s .ta k e n .a t h ig h c o n c e n tra tio n s o f the. a n t i b i o t i c .
T h e se d a ta w e re b r ie f ly re v ie w e d in th e in tro d u c tio n a n d w ill n o t b e r e ­
in tro d u c e d h e re e x c e p t fo r n e c e s s a r y c o m p a ris o n . The a u th o r b e lie v e s
t h a t th is is. a r e a s o n a b le a p p ro a c h b e c a u s e .th e a c tu a l m e c h a n ism o f CAP
a c tio n s h o u ld b e s t be d e te rm in e d from, r e s u lts o b ta in e d a t lo w c o n c e n tr a ­
tio n s w h ic h .are c o m p a tib le w ith CAP'S a c tio n in v i v o . T h is is. tru e
b e c a u s e th e re e x i s t s a v e r y r e a l p o s s i b i l i t y th a t the. o b s e r v e d e f f e c ts o f
CAP in h ig h c o n c e n tr a tio n s m ay w e ll b e s e c o n d a ry in n a t u r e .
The e v id e n c e a g a i n s t CAP in h ib itio n o f p e p tid y l t r a n s f e r a s e w a s
o b ta in e d a t high, c o n c e n tr a tio n s a s w e re th e r e s u lts fa v o rin g th is m e c h ­
a n ism o f a c ti o n . It h a s b e e n sh o w n th a t CAP in h ib its th e e x te n t.b u t n o t
th e r a te o f th e p u ro m y cin re a c tio n (3 4 ), th u s th e s e a u th o rs a rg u e th a t
42
• CAP does, .not in h ib it p e p tid y l tr a n s f e r a s e b u t so m e s te p prior to its
. a c ti o n . A lso i t h a s b e e n sh o w n t h a t CAP d o e s n o t in h ib it.th e ,a d d itio n .o f
ly s in e r e s id u e .to p o ly ly s y l-t- R N A .(3 2 ). T hus i t seem s, q u ite p ro b a b le
. t h a t CAP d o e s n o t in h ib it th e en zy m e d ire c tly , b u t m ay in te r f e r e w ith a
. p r o c e s s p rio r o r c o n c u rre n t to th e fo rm atio n , o f-th e p e p tid e , b o n d . T his is
.c o n s is te n t w ith .th e a b o v e p ro p o s a l t h a t CAP'S a c tio n on. p ro lin e p e p tid e
. s y n th e s is o c c u rs a t a n e a r ly s te p in p e p tid e s y n t h e s i s .
The, f i r s t p o s s i b i l i t y o f in h ib itio n , o f a s te p p rio r o r c o n c o m m ita n t
..to p e p tid e b o n d form ation, i s th e tr a n s l o c a s e s t e p . T h is p o s s i b i l i t y m ay
/ b e e lim in a te d due. to th e f a c t th a t th e form ation, o f th e d ip e p tid e is i n ­
h ib ite d , in th e p o ly C s y s te m . T his i s tru e b e c a u s e tr a n s lo c a tio n sh o u ld
n o t be re q u ire d fo r.th e. form ation, o f t'he f ir s t p e p tid e b o n d . T his a rg u m e n t
i s s u p p o rte d by th e f a c t th a t-th e . fo rm atio n , o f .d ip h e n y la la n in e from
p h e n y la la n y l-t-R N As d o e s n o t re q u ire the. p r e s e n c e o f th e G: fa c to r
( tr a n s lo c a s e fu n c tio n ) (6 0 ).
The se c o n d , s ta t e d p o s s ib ility , n a m e ly ,in h ib itio n o f rib o s o m a l m o v e­
m e n t, may. be. e lim in a te d ,f o r th e sam e, r e a s o n .a s th e f i r s t 's i n c e d ip e p tid e
fo rm atio n s h o u ld n o t re q u irb th e m o v em en t o f th e .r ib o s o m e . I n .f a c t , o n e
: m ig h t c o n s id e r rib o s o m a l m ovem en t and. tr a n s lo c a tio n to o c c u r s im u lta n e ­
o u s ly o r a c tu a lly be th e .v e r y sam e p r o c e s s .
The t h i r d .p o s s i b i l i t y , in h ib itio n o f th e e x p lu s io n o f d e a c y la te d
t-R N A , m ay be e lim in a te d by a s im ila r a rg u m e n t. If d e a c y la te d t-RNA i s
n o t r e le a s e d i t w ou ld , jam :-the d o n o r s i t e , b u t a p r e r e q u is ite fo r i t s
43
■p o s itio n .th e r e w o u ld be th e fo rm a tio n o f t h e .f i r s t p e p tid e b o n d w h ic h .is
in h ib ite d , b y CAP a s sh o w n , in ..the p o ly C s y s te m .
The fo u rth p o s s i b i l i t y , in h ib itio n .o f a m in o a c y l te rm in u s b in d in g o r
p ro p e r i n t e r a c t i o n , i s c o n s i s t e n t w ith th e r e s u lts from G A P's e f f e c t on
p ro lin e p e p tid e , s y n t h e s i s . A lso i t has. b e e n re c e n tly sh o w n b y P e s tk a
(22) t h a t CAP in h ib its th e b in d in g o f th e a m in o a c y l te rm in u s o f
a m in o a c y l-t-R N A to th e 508 s u b u n it o f the. rib o s o m e . He. o b ta in e d 50%
in h ib itio n .o f binding, a t 10 ^ M CAP. S in c e th e b a c te r ia l.r ib o s o m e h a s
b e e n sh o w n to p o s s e s s , tw o b in d in g s i t e s fo r a c y la te d t-R N A (6 0 ,6 1 ,6 2 ,
63), i t i s p o s s ib le th a t o n e s ite is m ore s e n s itiv e to b in d in g in h ib itio n
. th a n i s th e o th e r; P e s tk a 1s r e s u lts a t h ig h e r c o n c e n tr a tio n s s u p p o rt-th is
s u p p o s itio n s in c e , he o b s e r v e d 85% .in h ib itio n , o f b in d in g at. 10 ^M . CAP.
A lso p re v io u s s tu d ie s h a v e re p o rte d th a t CAP in h ib its , th e b in d in g o f
. ly s y l-t-R N A to rib o s o m e s to a s m a ll d e g re e . (10-15%) (15) a n d s tim u la te s
th e b in d in g o f N - a c e ty lp h e n y la la n y l-t-R N A to rib o s o m e s (6 4 ), b u t th e s e
- l a t t e r d a ta w e re found, to be. in e f f e c t e s p e c i a l l y a t lo n g in c u b a tio n
t i m e s . The p o s s i b i l i t y th a t CAP m a y ,in te r a c t a t tw o d iff e r e n t s i t e s is
. s u p p o rte d , b y th e f a c t -that CAP h a s b e e n sh o w n to b in d to th e 508 s u b u n it
a t tw o d is c r e te le v e ls , o f b in d in g (16). Thus i t w o u ld s e e m lik e ly from
t h e a b o v e o b s e r v a tio n s th a t CAP m ay a c t a t th e d o n o r o r a c c e p to r s i t e on
th e 508 s u b u n it in som e, m a n n er w h ic h in te r f e r e s w ith th e p ro p e r a t t a c h ­
m e n t o r in te r a c tio n o f th e a m in o a c y l- o r p e p tid y l-t-R N A w ith th e ir
r e s p e c tiv e , b in d in g s i t e s . The d a ta .f ro m .th e polyC. a re c o n s is te n t w ith
44
th is ty p e o f m o d el s in c e i t h a s sh o w n t h a t CAP in h ib its th e s y n th e s is o f
a l l th e p ro lin e p e p t i d e s T h i s s u g g e s ts , th a t CAP m ig h t a f f e c t th e ln itih
a tio n . o f a p ep tid e, chain, r a th e r th a n i t s e x te n s io n . T his m o d el is a ls o
s u p p o rte d .by th e r e s u l t s o b ta in e d . from- CAP1s e f f e c t on a n in v itro s y s te m
w h ich , u tiliz e s M S-2 RNA.t o ■produce, the. c o a t p ro te in o f th e p h a g e (2 7 ).
In .th e. M S-2 s y s te m th e p re s e n c e , o f CAP r e d u c e s th e le v e l o f c o m p le te d
a n d r e le a s e d c o a t p ro te in b u t d o e s, n o t g iv e r is e to p a r tia l p ro d u c ts r e ­
l e a s e d fro m .th e ; rib o s o m e . U pon a n a ly s is o f th e r e le a s e d c o a t p ro te in
v e r s u s th e rib o s o m a l b o und p e p tid e s i t was. fo u n d .th a t t h e r a tio o f b o und
to r e le a s e d p e p tid e s re m a in e d c o n s ta n t in -the p r e s e n c e o f v a rio u s c o n ­
c e n tr a tio n s o f CAP. T his in d ic a te s t h a t CAP d o e s n o t s to p p e p tid e c h a in
e lo n g a tio n th u s c a u s in g a p ile up, o f s m a ll p e p tid e s, on th e rib o so m e b u t
a c t s e a r lie r th a n th e e lo n g a tio n -.p ro c e s s ; th is is th e s a m e r e s u lt one o b ­
s e r v e s in .the. p o ly C s y s te m ; s m a ll p e p tid e s, a re n o t a c c u m u la te d . The
o n ly u n e x p la in e d e f f e c t o f CAP on th e fo rm a tio n .o f p ro lin e p e p tid e s is
th a t th e s y n th e s is o f th e s m a ll p e p tid e s is n o t in h ib ite d , to th e sam e e x ­
t e n t as. th e s y n th e s is o f th e la rg e p e p t i d e s . . It is- p o s s ib le , th a t th e
fo rm a tio n .o f th e s m a ll p e p tid e s is o c c u rrin g in som e u n u s u a l m an n er or
th a t th is p a rtic u la r, s te p i s n o t s e n s itiv e , to CAP fo r som e, o th e r r e a s o n .
O ne p o s s i b i l i t y w h ic h m ay e x p la in th is d if f e r e n tia l s e n s itiv ity , i s - th a t
GA P's a c tio n m ay o c c u r s p e c if ic a lly a t th e d o n o r s i t e in th e a re a o f th e
a c y l term in u s, o f th e p e p tid y l-t-R N A . If CAP'S a c tio n .o c c u rre d a t th e
a c c e p to r s i t e -then.-there w o u ld b e no d if f e r e n tia l e f f e c t e x p e c te d w h ic h
45
w o u ld d e p e n d .o n th e s i z e o f th e p ro d u c t.m a d e b e fo re i t s fo rm a tio n . But
i f CAP a c t s at. th e d o n o r s i t e i t s a c tio n , m ay a p p e a r to b e d if f e r e n tia l
a c c o rd in g to th e s i z e o f th e p e p tid e a tta c h e d to th e .t-RNA. Thus, fo r th e
fo rm a tio n o f th e f i r s t p e p tid e bond am inoac-yl-t-R 'N A m u s t a c t a s
p e p tid y l-t-R N A b u t i t m ay n o t i n te r a c t a t th e d o n o r s i t e in e x a c tly th e
sa m e m a n n er a s w o u ld p e p tid y l-t-R N A . The .sam e m ig h t b e tru e, o f th e
d ip e p tid y l-t-R N A a n d r e s u l t in. l e s s in h ib itio n o f its tr a n s f e r o f th e d i ­
p e p tid e , to th e a m in o a e y l-t-R N A in th e a c c e p to r s ite b y CAP. This
s u p p o s itio n i s s u p p o rte d b y th e f a c t t h a t in th e b a c te r ia l s y s te m a
N -b lo c k e d , a m in o a c y l-:t-RNA. i s re q u ire d to in itia te the. fo rm a tio n o f th e
f i r s t p e p tid e -b o n d (5 2 ,5 3 ,5 4 ,5 5 ,5 6 ,5 7 ,5 8 ) . T his re q u ire m e n t is o v erco m e
in th e s y n th e tic m e s s e n g e r sy ste m s.-b y u s in g m a g n esiu m io n .c o n c e n tr a ­
tio n s above.-10 mM. w h ic h a llo w s in itia tio n to o c c u r w ith a n u n b lo c k e d
a m in o a c y l-t-R N A .
I t is c o n c lu d e d t h a t , b a s e d o n CAP'S e ffe c t a t lo w c o n c e n tr a tio n s ,
CAP m ay a c t at. o r n e a r th e d o n o r s ite , a t w h ic h th e a c y l te rm in u s o f th e
p e p tid y l-t-R N A a tta c h e s , to th e 508 s u b u n it o f th e r ib o s o m e .. T h is -in te r­
a c tio n m ig h t p re v e n t th e p ro p e r a tta c h m e n t o f th e a c y l te rm in u s to. its
b in d in g s i t e . T his h y p o th e s is is c o n s i s t e n t w ith. the. d a ta o b ta in e d from
th e in h ib itio n o f th e p u fo m y cin r e a c tio n a t h ig h c o n c e n tr a tio n s o f CAP.
If CAP -in h ib its a n y s te p p rio r .to,-the fo rm atio n o f p e p tid y lp u ro m y c in o r a
p e p t i d e , th e form ation, o f th e p e p tid e b o n d w ill a p p e a r .to b e in h ib ite d
s i n c e : i t i s .t h e o n ly s te p a s s a y e d . A lso from s tu d ie s - o n th e p u ro m y cin
46
r e a c tio n w ith rib o s o m a l b o und n a s c e n t p e p tid e s is o la te d from, grow ing
E_. coH (34) i t is s u g g e s te d t h a t p e p tid y l-t-R N A m ay e x i s t in th re e s t a t e s
on th e rib o so m e : (a) a s i t e a t w h ic h th e p e p tid y l-t-R N A is r e s i s t a n t to
th e a c tio n o f GAP in th e a b s e n c e o f GTP a n d .s u p e r n a ta n t fa c to rs ; (b) a
s i t e a t w h ic h th e p e p tid y l-t-R N A i s s e n s itiv e , to th e a c tio n o f CAP in th e
a b s e n c e , o f GTP a n d s u p e r n a ta n t f a c to r s ; and. (c) a s i t e a t w h ic h th e
p e p tid y l-t-R N A i s s e n s itiv e , to th e a c tio n of-CAP in th e p r e s e n c e o f GTP
a n d s u p e r n a ta n t f a c t o r s . T h ese, s i t e s m ay c o r r e s p o n d .r e s p e c tiv e ly to:
(a) th e d o n o r s ite ; .(b) so m e s ite , in te rm e d ia te b e tw e e n the. d o n o r an d a c ­
c e p to r s i t e s ; (c) th e a c c e p to r s i t e . If th is h y p o th e s is i s tru e then, i t is
o b v io u s t h a t CAP'S a c tio n o c c u rs a t th e d o n o r s i t e .
C o n s id e r -the th re e
s i t e s a s p ro p o se d : (a) i f th e p e p tid y l-t-R N A is a lre a d y , in th e d o n o r s ite
i t s h o u ld b e im m une to th e a c tio n o f CAP a s i s o b serv ed '; (b) if th e
p e p tid y l-t-R N A h a s n o t e n te r e d th e d o n o r s i t e i t sh o u ld b e s e n s itiv e to
th e a c tio n o f CAP a s o b s e rv e d ; a n d (c) a s in ,th e c a s e o f (b) th e p e p tid y lt-RN A m u s t be m oved to th e d o n o r s i t e and. th u s b e s e n s i t i v e to th e
a c tio n o f CAP a s o b s e r v e d . T his w ork w a s c o n d u c te d a t a CAP c o n c e n ­
tr a tio n o f 100 (ig/m l b u t th e au th o rs, s t a t e t h a t th e o b s e r v e d e f f e c t o f CAP
oh th e purom ycin. r e a c tio n d o e s h o t s ig n if ic a n tly v a ry i f th e CAP c o n c e n ­
tra tio n is v a r ie d b e tw e e n 20 a n d 500 p g / m l.
The d iffe re n c e s , b e tw e e n the b a c te r ia l an d m am m alian p ro te in s y n ­
th e s i z in g s y s te m s a ls o g iv e a good in d ic a tio n .th a t CAP'S a c tio n o c c u rs
a t th e d o n o r s i t e . O ne o f th e m a jo r d if f e r e n c e s b e tw e e n .th e s e tw o
47
s y ste m s , a p p e a rs to .in v o lv e the. d o n o r s i t e . In .the c e l l - f r e e s y s te m d e ­
riv e d from r a b b it r e tic u lo c y te s i t h a s b e e n sh o w n t h a t d e a c y la te d t-R N A
a p p e a rs .to b in d a t -the d o n o r s i t e (65) b u t n e ith e r N - a c e ty lp h e n y la la n y l.t-R N A (65) n o r p h e n y la la n y l-t-R N A .(6 5 ) a p p e a rs to b in d d ir e c tly to th e
d o n o r s i t e . T h e se s tu d ie s a l s o in d ic a te th a t ,the. r e tic u lo c y te s y s te m h a s
a s tr in g e n t re q u ire m e n t fo r d e a c y la te d .t-RNA for th e in itia tio n o f p o ly ­
p h e n y la la n in e s y n th e s is (65) . The b a c te r ia lly d e riv e d rib o s o m a l s y s te m
. a p p e a rs to d iffe r i n .a l l th e a b o v e r e s p e c t s . The b a c te r ia l rib o s o m e .(70S)
w ill b in d d e a c y la te d t-R N A (67,6 8 ) b u t n o -re q u ire m e n t fo r i t in .in itia tio n
h a s b e e n d e m o n s tr a te d , Also, i t a p p e a rs, th a t a m in o a c y l-t-R N A is b o und
to b o th th e d o n o r a n d .a c c e p to r s i t e s s in c e , d ip e p tid e fo rm a tio n o c c u rs on
th e rib o so m e in th e p r e s e n c e o f o n ly b in d in g fa c to rs (T a n d GTP) w ith o u t
a d d e d tr a n s l o c a s e (G) (6 0 ). A nother s tu d y h a s sh o w n th a t
N -a c e ty lp h e n y la la n y l-t-R N A is. b o u n d d ir e c tly to th e d o n o r s i t e (58),.
■It has. b e e n sh o w n th a t CAP d o e s n o t in h ib it c e l l - f r e e s y n th e s is , in
. s y ste m s , d e riv e d from, m am m als (80S. rib o s o m e s) (6 9 ,7 0 ) b u t v a rio u s
a u th o rs h a v e re p o rte d , th a t i t d id in h ib it p ro te in s y n th e s is in c e ll - f r e e
s y s te m s , d e riv e d from r e tic u lo c y te s (.71, 7 2 ). H o w ev er th e m e ch a n ism o f
a c tio n , o f CAP in .th is , s y s te m a p p e a rs, to -b e d iffe re n t th a n t h a t in. th e 70S
. rib o s o m a l s y s te m s in c e i t w a s o b s e r v e d .th a t CAP in h ib ite d th e b in d in g o f
p o lyU to re tic u lo c y te .r ib o s o m e s . (7 3 ).
B ase d on. th e d iffe re n c e s , b e tw e e n .th e 70S an d 8 0 S .rib o s o m a l s y s ­
te m s CAP'S a c tio n .m a y o c c u r a t th e d o n o r s i t e o f th e 70S rib o so m e s in c e
48
i t a p p e a rs th a t th is s i t e o r in te r a c tio n a t th is s i t e ,is o n e o f th e m ajo r
d if f e r e n c e s b e tw e e n th e tw o s y s t e m s .
The e x a c t m e c h a n ism o f GAP's a c tio n , a t th e d o n o r s i t e may- b e e x ­
tr e m e ly d if f ic u lt to e lu c id a te u n le s s te c h n iq u e s are. d e v e lo p e d to s p e c i ­
f ic a lly e x a m in e th e in d iv id u a l s te p s in v o lv e d i n the. s y n th e s is o f
p r o t e i n s . S uch s tu d ie s m ay be a c c o m p lis h e d e ith e r b y s p e c if ic a lly i n ­
h ib itin g s e le c te d s te p s in. p ro te in s y n th e s is o r b y f r a c tio n a tin g th e p ro ­
te in s y n th e s iz in g s y s te m to a s u f f ic ie n t d e g re e to a llo w e x a m in a tio n o f
th e s p e c if ic s te p s , in d e p e n d e n t o f th e o t h e r s . P ro c e d u re s o f th e la t t e r
type: h a v e b e e n .r e c e n tly d e v e lo p e d .for c e l l - f r e e p ro te in s y n th e s iz in g
s y s te m s d e riv e d .fro m r e tic u lo c y te s (66) a n d .fro m E . c o li (7 4 ).
The e x tre m e s e n s i t i v i t y o f the. p o ly C s y s te m to CAP s u g g e s ts th a t
fu rth e r s tu d ie s be u n d e rta k e n on GAP’s e f f e c t on the. s y s te m a n d p erm u ­
ta tio n s . o f i t . I t w o u ld b e o f in t e r e s t to s tu d y the. p u ro m y cin r e a c tio n in
th e p o ly C s y s te m a n d GAP’s e f f e c t upon, i t .
LITERATURE CITED
1 . W is s e m a n , C . L ., R E . H a h a ii, H„ H o p p s , and. J 6 E . S m a d e l,
F e d e ra tio n P r o c . 1 2 ,4 6 6 .(1 9 5 3 ).
2 . W is s e m a n , C . L ., J . E. S m a d e l, F. E . H a h n , a n d H . E . H o p p s z J .
. B a c t. .67, 662 (1 9 5 4 ).
3 . G a l e z E . F., a n d I . P. P o lic e s, B io c h e m .J . 5 3 ,4 9 3 (1 9 5 3 ).
4 . D e M o s s , J . A . , and. G . D . N o v e lli , B io c h im . e t B io p h y s . A cta 1 8 ,
5 . L a c k s z S ., a n d F. G r o s j I . M o l. B io l. I , 301 (1 9 5 9 ).
6 . N ire n b e rg , M . W ., a n d J . H . M a t t h a e i ,. P r o c . N a t l . A c a d . S c i . U . S .
47, 497 (1 9 6 1 ).
7 . N a th a n s , D ., a n d .F. L ip m a n n , P r o c . N a tl. A cad. S c i . U . S . 47, 497
(1 9 6 1 ).
8 . R e n d i, R , an d S. O c h o a , I . B io l. C h e m . 237, 3711 (19 6 2 ).
9 . K u c a n , Z ., a n d F. L ip m arm , I . B io l. C h e m . 239 , 516, (1 9 6 4 ).
I. 0 . S o , A. G ., a n d E . W. D a v ie , B io c h e m istry J , 132 (1 9 6 3 ).
I I . V a zq u e z , D . , B io c h e m . B iophys . R e s e a rc h Q om m un. 12 , 469, (19 63).
1 2. V azq u ez , D . , B io c h em . B iophys . R e s e a rc h C om m un. 1_5 , 262 (1 9 6 4 ).
13 .■ W o lfe ,A . D ., a n d F-. E . H a h n , F e d e r a tio n , P ro c . 23 , 269 (1 9 6 4 ).
1 4 . V a z q u e z , D ., B io c h im . e t B io p h y s . A cta 114, 289 (1 9 6 5 ).
1 5. W o lfe , A. D ., a n d F. E . H a h n , B io c h im . e t . B iophys . A cta 95 , 146
(1 9 6 5 ).
1 6. D a s , H ., A. G o ld s te in , a n d L. H anner,.. J. M o l. P h a rm .
158 (1 9 6 6 ).
50
1 7 . Speyer., J . F ., P. L e n g y e l, C . B a s ilio , A . J . W a h b a , R. S . G arsiper,
a n d S . O c h o a , C o ld -S p rin g H arb o r S y m p o sia Q u a n t. B io l. 2 8 , 559 (1963)„
1 8. J a r d e s t s k y , O ., a n d G . R. J u lia n , N a tu re 2 0 1 , 396 (1 9 6 4 ).
1 9 . N a k a m o to , T., T. W. C o n w a y , J . E. A lle n d e , G . J . S p y rid e s , an d
F. L ip m a n n , C o ld S pring H arb o r S y m p o sia Q u a n t. B io l. 2 8 , 227 (1 9 6 3 ).
2.0. S u a r e z , G .,. and. D . N a th a n s , B io c h e m . B io p h y s . R e s e a rc h C om m un.
1 8 , 743 (1 9 6 5 ).
2 1 . -V a z q u e z , I ) ., a n d R. E. M onro , B io c h im . e t B io p h y s . A cta 1 4 5 , 155
(1 9 6 7 ).
22 .. P e s t k a > S ., B io c h em . B iophys . R e s e a rc h C om m un. 3 6 , 589 (1 9 6 9 ).
2 3 . O fe n g a n d , J .,. a n d R. H a s e lk o r n , B io c h em . B io p h y s . R e s e a rc h
C om m un. J 5 ,469 (1 9 6 2 ).
2 4 . V a z q u e z , D ., B io c h im . e t B io p h y s . A cta 114, 289 (1 9 6 6 ).
2 5 . J u lia n , G . R ., J . M o l. B io l. 12., 9 (1 9 6 5 ).
2 6 . N a th a n s , D .,. J . M o l. B io l. 13 , 521 (1 9 6 5 ).
2 7 . M o rg a n , E . J ., a n d G . R. J u lia n , P e rs o n a l c o m m u n ic a tio n (1 9 6 9 ).
2 8 . S m ith , J . D ., R . R . T r a u t, G . M . B la c k b u rn , an d R . E . M o n ro , J . M o l.
. B io l. 13., 617 (1 9 6 5 ).
2 9 . T r a u t, R .R ., a n d R iE . M o n ro , J . M o l. B io l. 1 0 , 63 (1 9 6 4 ).
3 0 . N a th a n s , D ., P r o c . N a tl. A c ad . S c i . U . S . 5 1 , 585 (1 9 6 4 ).
3 1 . C u n d lif f e , E ., a n d K. M c Q u ille n , J . M o l. B iol. 3 0 , 137 (1 9 6 7 ).
3 2 . G o tte s m a n , M . E ., J . B io l. C h e m . 2 4 2 ,5 5 6 4 (1 9 6 7 ).
3 3 . C a n n o n , M ., E uropean. J , B io c h em . 7, 137 (1 9 6 8 ).
51
34 „ W e b e r, M . J„, and. To A a D e M o s s , I . B a c t, 97, 1099 (1 9 6 9 ),
3 5 . W a h b a , A.. J . , R. S . G a rd n e r, C . B a s illo , R. S , M ille r , J . F. S p e y e r,
a n d P. L e n g e l, F re e . N a tl. A cad . Sci.. U . S . '49 , 116, (1 9 6 3 ).
3.6. S p e y e r, J . F ., P . L e n y g e l, C . B a s ilio , an d S . O c h o a , P r o c . N a t).
A c a d . S c i . U . S . 4 8 , 63 (1 9 6 2 ).
3 7 . H a s e lk o r n , R ., and. V. A. F rie d , P r o c . N a tl. A c ad . S c i . U . S . 5 1 , 308
(1 9 6 4 ).
3 8 . H a s e lk o r n , R ., a n d V. A. F rie d , P r o c . N a t l . A c a d S c i . U . S . .5 1 , 1000
(1 9 6 4 ).
3 9 . G o ld b e rg , I . H„, a n d K. M its u g i,.-■B io c h e m istry 6_, 372 (1 9 6 7 ).
4 0 . M o o re , P., T. M o l. B io l. 1 8 , 8 (.19.66).
4 1 . T a n a k a , N ., K. S a s .h ik a ta , H ..Y a m a g u c h i, an d H . U m e z a w a , %.
B io c h em . 60 , 405 (19 66 ).
42 . T a n a k a , N ., K. S a s h i k a t a , an d H . U m ezaw a,. T. A n tib io tic s , Sen A.
2 0 , 115,(1967).
43 . B h u y a n , B. K ., B io c h e m ic a l P h a rm a c o lo g y 1 6 , 1411 (1 9 6 7 ).
4 4 . M a o ,T . C . H ., T. B a c te r io l, 9 4 , 800 (.1967).
4 5 . Y a m a g u c h i, H ., a n d N . T a n a k a , I . B io c h e m . 60 , 632. (1 9 6 6 ).
4 6 . Y a m a g u c h i, H„, Y. Y o s h id a , an d N . T a n a k a , I . B io c h e m . 6 0 , 246
(1 9 6 6 ).
4 7 . S m ith , I . D ., I . M o l. B io l. 8 , 772 (1 9 6 4 ).
4 8 . S m ith , M . A ., a n d M . A. S ta h m a n n , B io c h em . B io p h y s . R e s e a rc h
. C om m un. 13 , 251 (1 9 6 3 ).
52
. 4 9 . S c h w a r tz , D . P., a n d M . J . P a l l a n s c h f A n al. C h e m . 3 0 ,2 1 9 .(1 9 5 8 ).
5 0 . S c h a e c h te r , M ., J . Mol-. B io l. 7, 561 (1 9 6 8 ).
5 1 . S c h l e s s l n q e r , D ., I . M o l. B io l. 7, 569 (1 9 6 3 ).
5 2 . C la r k , B. F. C ., a n d K. A. M a rc h e r, I , M o l. B io l. 17, 394 (1 9 6 6 ).
5 3 . A dam s,. J . M ., a n d M . R. C a p e c c h i, P r o c . N a t l . A c a d . S e i . U . S . 55 ,
147 (1 9 6 6 ).
5 4 . W e b s te r , R . E . , D . L. E n g e lh a rd t, a n d N . D . ,Z in d er, P r o c . N a tl.
A c ad . S c i . U . S . 5 5 , 155 ,(1966).
5 5 . N o ll, H ., S c ie n c e . 1 5 1 , 1241 (1 9 6 6 ).
5 6 . B r e ts c h e r , M . S ., C o ld S p rin g H a rb o r S y m p o sia Q u a n t. B io l. 3 1 ,2 8 9
(1 9 6 6 ) .
5 7 . L e d e r , P., a n d H . B u rs z ty n , C o ld 1 S p rin g H arbpr S y m p o sia Q u a n t.
B io l. 3 1 ,2 9 7 (1 9 6 6 ).
5 8 . L u c a s - L e n a r d , J ., a n d F. L ip m a n n , P r o c . N a tl. A c ad . S c i . 57, 1050
(1 9 6 7 ) .
5 9 . M c L e a n , I . W ., J . L . S c h w a b , A. B. H i l l e g a s , a n d A. S . S c h lin g m a n ,
I . C lin . I n v e s t. 1 8 , 953 (1 9 4 9 ).
6 0 . L u c a s - L e n a r d f J .,. an d A. H a e n n i, P r o c . N a t l . A c a d . S c i . U . S . 59 ,
554 (1 9 6 8 ).
6 1 . E r b e f R. W ., M . M . N a u , P. L e d e r,. I . M o l. B io l. 3 8 ,4 4 1 (1 9 6 9 ).
62 .. W a ts o n , J . D ., B u ll. S o c . C h im . B io l. 4 6 , 1399. (1 9 6 4 ).
6 3 . R a v e l, J . M ., R. L . S h o re y , S . F r o e h n e r, and.W . S h iv e , A rc h .
B io c h em . B io p h y s . 1.25 , 514 (1 9 6 8 ).
53
.64. H e r n e r , A. E ., I . H . G o ld b e rg , a n d L , 5 . C o h e n , B io c h e m istry 8 ,
1335 (19.69).
6 5 . H a r d e s ty , B ., W. C u lp , a n d W. M c K e e h a n , C o ld S p rin g H arbor
S y m p o sia Q u a n t. B io l., in p r e s s (1 9 6 9 ).
6 6 . M o s t e l l e r 1 R ., W. C u lp , an d B. H a r d e s ty , B io c h em . B io p h y s .
R e s e a rc h C om m un. 3 0 , 631 (1 9 6 8 ).
6 7 . T a k a n a m i, M .„ B io c h e m . e t B io p h y s . A cta 6 l , 432 (1 9 6 2 ).
6 8 . C a n n o n 1 M ., R. K rug, a n d W. G ilb e r t, T. M o l. B io l. 7, 360 (1 9 6 3 ).
6 9 . O c h o a 1J r ., a n d I . B. W e i n s t e i n , T. B ib l. C h e m . 239 , 3834 (1 9 6 4 ).
70.. K rug, R ., a s c ite d , by. K ucan a n d L ip m a n n , o p . c i t . , p . 519.
7 1 . W e is b e r g e r , A. S ., S . A rm e n tro u t, a n d S . W o lf e , P r o c . N a tl, A c a d .
S c i . 5 0 ,8 6 .( 1 9 6 3 ) .
7 2 . W e is b e r g e r , A. S ., S . W o lf e , a n d S . A rm e n tro u t, I , E x p tl. M e d . 1 2 0 ,
1 6 1 .(1 9 6 4 ).
7 3 . W e is b e r g e r , A i S ., a n d S . W o l f e F e d e ra tio n P r o c . 2 3 , 967 (1 9 6 6 ).
7 4 . L ip m a n n 1 F., S c ie n c e 1 6 4 , 1024 (1 9 6 9 ).
M ON TA N A S T A T E U N IV E R SIT Y L IB R A R IE S
3 1762
0005652 O
0378
Ir9
cop.2
Irvin, James D.
The effect of ehloram
phenicol on the in
vitro synthesis...
MAMK A N Q A O P W K g g
9
1979
I /.X
/f-,>
Jz-f
Oo^>.cS