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Studies On The Enzymatic Formation Of Amino Acid - Ribonucleic Acid Compounds

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Studies On The Enzymatic Formation Of Amino Acid - Ribonucleic Acid Compounds

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STUDIES O N THE ENZYMATIC FORMATION OF
AMINO ACID-RIBONUCLEIC ACID COMPOUNDS
by
E s th e r Haney A lle n
A D i s s e r t a t i o n P re s e n te d to th e
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u l f i l l m e n t o f th e
R eq u irem en ts f o r th e Degree.
DOCTOR OF PHILOSOPHY
(B io c h e m istry and N u t r i ti o n )
June I960
UNIVERSITY O F SO U T H E R N CALIFORNIA
GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES 7, CALIFORNIA
This dissertation, written by
.A ?* ?-* ®
under the direction of h$T....Dissertation C om
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R O F P H I L O S O P H Y
Dean
D ate...... .June.... 19.6.0..
DISSERTATION COMMITTEE
ACKNOWLEDGEMENTS
I w i s h 't o th a n k D r. P a u l S altm an f o r h i s c o n ti n
uous enco u rag em en t and i n t e r e s t i n t h i s w ork, and f o r
s e c u rin g th e n e c e s s a r y s u p p o rt f o r th e e a r l y p a r t o f
th e s e s t u d i e s . I am g r a t e f u l , a l s o , to D r. John Mehl f o r
h i s g u id a n c e i n my g r a d u a te program and f o r h i s c o n fid e n c e
i n e n c o u ra g in g me to c a r r y o u t t h i s r e s e a r c h p ro b lem .
A s p e c i a l word o f g r a t i t u d e i s e x te n d e d to D r.
R ic h a rd Schw eet who h a s g u id ed th e p r o g r e s s io n o f th e
r e s e a r c h s t u d i e s . He h as a llo w e d me to p u rsu e t h i s i n
v e s t i g a t i o n i n h i s l a b o r a t o r y a t th e C ity o f Hope M edical
C e n te r , and th e a s s o c i a t i o n w ith him a n d - h is r e s e a r c h
s t a f f h a s b een an in v a l u a b l e e x p e r ie n c e .
D o c to rs John Webb, W a lte r Marx, Donald V i s s e r ,
and Norman K h arasch have a l s o a id e d me g r e a t l y by s e rv in g
on my d o c t o r a l com m ittee and by t h e i r i n t e r e s t e v id e n c e d
i n v a lu a b le d i s c u s s i o n s o f d i f f e r e n t a s p e c t s o f my
r e s e a r c h p r o p o s i t i o n s and d i s s e r t a t i o n s tu d y .
I am in d e b te d to th e N a tio n a l C ancer I n s t i t u t e ,
U n ite d S t a t e s P u b lic H e a lth S e r v ic e , f o r th e p r o v i s i o n o f
a P r e d o c to r a l R e se a rc h F e llo w s h ip d u rin g th e l a s t two
y e a r s o f my g ra d u a te s tu d y .
A s p e c i a l o f f e r o f th a n k s goes to my h u sb an d ,
P a u l, whose c o n fid e n c e and encouragem ent th ro u g h o u t my
y e a r s o f g ra d u a te s tu d y have made th e m ost d i f f i c u l t
a s p e c t s o f t h i s e n d ea v o r much more p l e a s a n t .
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS .......................................................................... i i
LIST OF TABLES ..................................................... v
LIST OF ILLUSTRATIONS................................................................ v i
I . HISTORICAL INTRODUCTION ............................................ 1
N ature o f th e P r o t e i n M olecule
S t r u c t u r a l s p e c i f i c i t y
P r o t e in p r e c u r s o r s
S tu d ie s o f P r o t e in S y n th e s is in
C e ll- F r e e P r e p a r a tio n s
C h a r a c t e r i s t i c s o f c e l l - f r e e system s
E nergy so u rc e f o r p e p tid e bond
fo rm a tio n
A c tiv a te d I n te r m e d ia te s i n P r o t e in
S y n th e s is
Amino a c id a c t i v a t i o n
Amino a c i d - r i b o n u c l e i c a c id
fo rm a tio n
I I . STATEMENT OF THE PROBLEM AND PLAN OF
A T T A C K ......................................................................... . 15
I I I . MATERIALS AND METHODS . . ................................... 17
M a te r ia ls
Anim als
C hem icals
I s o to p ic compounds
O ther m a t e r i a l s
Methods
A ssay p ro c e d u re s
P y ro p h o sp h ate exchange a s s a y
Amino a c id i n c o r p o r a ti o n i n to r i b o
n u c le ic a c id
ATP in c o r p o r a ti o n i n t o r i b o n u c l e i c
a c id
Amino a c id in c o r p o r a ti o n i n to
p r o t e i n
iii
Page
A n a l y ti c a l m ethods
Enzyme p r e p a r a t i o n
P r e p a r a t io n o f pH 5 enzymes
F r a c t i o n a t i o n o f pH 5 enzymes
Ribosome p r e p a r a t i o n from r a b b i t
r e t i c u l o c y t e s
P r e p a r a t io n o f enzyme f r a c t i o n from
r a b b i t r e t i c u l o c y t e s
R ib o n u c le ic a c id p r e p a r a t i o n
S tu d ie s o f Amino Acid A c tiv a tio n
S u b s tr a te s o f pH 5 enzymes
S t a b i l i t y o f pH 5 enzymes
P r o p e r t ie s o f pH 5 enzymes
I n h i b i t i o n o f enzyme a c t i v i t y
Enzyme f r a c t i o n a t i o n and p u r i f i c a t i o n
I n c o r p o r a tio n o f Amino A cids i n t o
R ib o n u c le ic Acid
The r o l e o f a c t i v a t i n g enzymes
Assay c o n d itio n s
Amino a c id t r a n s f e r r i b o n u c l e i c a c id
S p e c i f i c i t y o f amino a c i d - r i b o n u c l e i c
a c id f o rm a tio n
P r o p e r t i e s o f t r a n s f e r r i b o n u c l e i c
a c id
S tu d ie s o f th e amino a c i d - r i b o n u c l e i c
a c id lin k a g e
E f f e c t s o f i n h i b i t o r s on amino a c i d -
r i b o n u c l e i c a c id fo rm a tio n
Amino A c id -R ib o n u c le ic A cid F o rm atio n
a s an In te r m e d ia te S tep i n P r o t e in
S y n th e s is
The s y n th e s is o f hem oglobin i n a
c e l l - f r e e system
P r o p e r t ie s o f th e amino a c id in c o rp o
r a t i o n system
The r o l e o f t r a n s f e r r i b o n u c l e i c a c id
i n hem oglobin s y n th e s is
IV. EXPERIMENTAL RESULTS
3*+
V. DISCUSSION
93
V I. SUM M ARY 108
LITERATURE CITED
113
iv
LIST OF TABLES
Table Page
1 . Amino A cid A c t iv a ti o n by pH 5 Enzymes . . . 35
2 . S t a b i l i t y o f A c tiv a tin g Enzymes .......................... 38
3 . E f f e c t o f S u b s tr a te s on I n h i b i t i o n by
E -C h lo ro m e rc u rib e n z o a te ........................................ ^6
*+. F r a c t i o n a t i o n o f pH 5 E n z y m e s ............................... *+ 7
5. Amino A cid I n c o r p o r a tio n i n t o R ib o n u c le ic
A cid ................................................................................... 50
6 . F r a c t i o n a t i o n o f R ib o n u c le ic A c i d ..................... 61
7 . S p e c if ic A c t i v i t y o f Amino A c id -R ib o n u c le ic
A cid Compounds ........................................................... 62
8. C ^-A T P and C ^-A m ino Acid I n c o r p o r a tio n
i n to P re in c u b a te d R ib o n u c le ic Acid . . . . 72
9 . I n c o r p o r a tio n o f C ^ - L e u c in e i n to P r o t e in . 82
10. E f f e c t o f R ib o n u c le ic Acid on C ^ -L e u c in e
I n c o r p o r a tio n i n t o P r o te in .............................. 87
11. T r a n s f e r o f Amino A cids from R ib o n u c le ic
A cid to P r o t e i n ........................................................... 91
v
LIST OF ILLUSTRATIONS
F ig u re Page
1 . F r a c t i o n a t i o n Scheme f o r pH 5 Enzymes . . . . 25
2 . E f f e c t o f ATP C o n c e n tr a tio n on L eucine
A c t iv a ti o n .......................................................................... *+1
3. E f f e c t o f L eucine C o n c e n tra tio n on
L eucine A c t iv a ti o n ...................................................... *+2
k. I n h i b i t i o n o f pH 5 Enzymes by £ -
C h lo ro m e rc u rib e n z o a te (PCMB) ................................... M +
5. E f f e c t o f G lu ta th io n e on Leucine-RNA
F o rm a tio n a t Two L ev e ls o f A c tiv a tin g
Enzyme .................................................................................... 52
6 . E f f e c t o f A c tiv a tin g Enzyme C o n c e n tra tio n
on Leucine-RNA F o rm a tio n ........................................ 53
7 . E f f e c t o f C ^-A m ino Acid C o n c e n tra tio n on
Amino Acid-RNA F o rm atio n ................................... 55
8 . E f f e c t o f ATP C o n c e n tra tio n on Leucine-RNA
F o r m a t i o n ............................................................................... 58
9 . Time C ourse o f Leucine-RNA F o rm atio n ..................... 58
10. E f f e c t o f RNA C o n c e n tra tio n on Leucine-RNA
F o r m a t i o n .............................................................................. 59
11. R ate o f Leucine-RNA F o rm a tio n w ith T r a n s f e r
RNA and E . c o l i R N A ...................................................... 66
12. A b s o rp tio n Spectrum o f T r a n s f e r RNA B efo re
and A f te r H y d ro ly s is ................................................. 68
13. E f f e c t o f AM P on Threonine-RNA F o rm a tio n . . . 76
vi
I . HISTORICAL INTRODUCTION
Twenty y e a rs a g o , b io c h e m is ts r e p o r t e d t h a t th e
mechanism o f th e s y n t h e s i s o f p r o t e i n i n th e l i v i n g c e l l
was v e ry o b sc u re and c o u ld be c l a r i f i e d o n ly th ro u g h th e
stu d y o f c o u p le d r e a c t i o n s i n th e m e ta b o lic sy stem . The
e s s e n t i a l t a s k b e fo re th e i n v e s t i g a t o r s i n p r o t e i n
s y n th e s is was t o d is c o v e r how th e c e l l p u ts to g e th e r in
p e p tid e lin k a g e tw en ty amino a c id s i n th e o b se rv ed v a r i e t y
o f s p e c i f i c , g e n e t i c a l l y d e te rm in e d s e q u e n c e s . E a r ly
w orkers s tr u g g le d w ith b e w ild e r in g ly com plex system s i n
w hich g r e a t s a t i s f a c t i o n was d e riv e d from f in d in g t h a t an
amino a c id r e a l l y found i t s way i n to p r o t e i n a t a l l . The
in te r m e d ia te r e a c t i o n s in v o lv e d have o n ly r e c e n t l y become
a p p ro a c h a b le a s c h em ical p ro b lem s, th u s c a u sin g an un
p re c e d e n te d grow th o f i n t e r e s t i n p r o t e i n s y n th e s is among
b io c h e m is ts . T h is sudden upswing o f i n t e r e s t has con
t r i b u t e d a g r e a t v a r i e t y o f t h e o r e t i c a l and e x p e rim e n ta l
a p p ro a ch e s to th e r a p i d l y expanding l i t e r a t u r e on p r o t e i n
s y n t h e s i s . A lthough th e g r e a t e s t d i s c o v e r i e s s t i l l l i e
b e fo re u s , q u i te enough h a s been a lr e a d y le a r n e d to make
w o rth w h ile a summary o f th e p r e s e n t p o s i t i o n .
1
Nature of the Protein Molecule
S t r u c t u r a l s p e c i f i c i t y
A p r o t e i n m o lecu le I s g e n e r a l l y re g a rd e d a s a
long c h a in o f amino a c id s u n ite d by a lp h a - p e p tid e bonds
and s u b j e c t to v a r io u s k in d s o f se co n d a ry b o n d in g . From
r e c e n t work on th e s t r u c t u r e o f such p r o t e i n s a s i n s u l i n ,
r i b o n u c l e a s e , and a d r e n o c o r tic o tr o p h ic horm one, i t ap
p e a rs t h a t th e s t r u c t u r e i s c o m p le te ly s p e c i f i c . A g iv e n
p o s i t i o n i n th e c h a in can "be o c c u p ie d by o n ly one o f th e
tw en ty n a t u r a l l y o c c u r rin g L-amino a c i d s . T h is i s p a r
t i c u l a r l y c l e a r when one o b se rv e s t h a t th e e s t a b l i s h e d
seq u en ces f o r r e l a t e d p r o t e i n s from d i f f e r e n t s p e c ie s
( i . e . , i n s u l i n s ) a re i d e n t i c a l e x c e p t f o r a v e ry few
s u b s t i t u t i o n s o f one amino a c id f o r a n o th e r ( 1 ) . Such a
b ro ad s ta te m e n t m ust be q u a l i f i e d by n o tin g t h a t th e
te c h n iq u e s used i n s t r u c t u r e d e te r m in a tio n would n o t
d e t e c t o c c a s io n a l d e v ia ti o n s from a p re d o m in a n tly unique
s t r u c t u r e . I t h as been found t h a t c e r t a i n u n n a tu r a l
amino a c i d s such a s e t h i o n i n e , n o r l e u c in e , and p - f l u o r o -
p h e n y la la n in e -can'be in c o r p o r a te d i n t o p r o t e i n ( 2 - b ) . A
q u a n t i t a t i v e e x a m in a tio n , how ever, r e v e a l s th e g r e a t
s e l e c t i v i t y o f th e p r o t e i n s y n th e s iz in g m echanism .
E th io n in e i s a v e ry poor s u b s t i t u t e f o r m e th io n in e , and
th e o b v io u s in f e r e n c e i s t h a t l e s s c l o s e l y r e l a t e d amino
a c i d s a r e even p o o re r s u b s t i t u t e s . Such a view o f p r o t e i n
s y n th e s is a s a h ig h ly p r e c i s e mechanism le a d in g t o a
unique sequence o f amino a c id s h as a ls o been d e riv e d from
n u t r i t i o n a l e x p e rim e n ts such a s th o s e o f G e ig e r (5 ) and
Cannon (6 ) who d e m o n stra te d t h a t a l l th e n e c e s s a r y amino
a c id s m ust be s im u lta n e o u s ly p r e s e n t i n o r d e r to e f f e c t
n e t p r o t e i n s y n t h e s i s .
P r o t e in p r e c u r s o r s
The th e o r y o f p r o t e i n s y n th e s is t h a t g a in e d m ost
ILl
fa v o r d u rin g th e p re - c a rb o n p e rio d was t h a t p r o t e i n
s y n th e s is was m ed ia te d by p r o t e o l y t i c enzym es. E a r ly
c a l c u l a t i o n s by B orsook and D ubnoff (7) a s w e ll a s sub
se q u e n t e x p e rim e n ta l o b s e r v a t i o n s , have shown t h a t p e p tid e
bond fo rm a tio n i s e n d e rg o n ic w ith a s ta n d a rd f r e e en erg y
in c r e a s e o f *+00 to 3000 c a l o r i e s p e r m ole. In e v e ry case
th e e q u ilib r iu m p o in t o f th e r e a c t i o n i s overw helm ingly
tow ard h y d r o ly s is r a t h e r th a n s y n t h e s i s . F u rth e rm o re , th e
p r o t e o l y t i c enzymes s tu d ie d th u s f a r do n o t have th e s id e
c h a in s p e c i f i c i t y a d eq u a te to p ro v id e a p r e c i s e l y o rd e re d
m o le c u le . A lthough many i n t e r e s t i n g o b s e r v a tio n s have
been made w ith p r o t e o l y t i c enzym es, a c lo s e r e l a t i o n s h i p
to p r o t e i n s y n th e s is seems d o u b tf u l.
The c l a s s i c s t u d i e s o f Schoenheim er and h i s
a s s o c i a t e s (8) i n t e r e s t e d b io c h e m is ts i n th e e x te n s iv e
f l u x betw een amino a c id s and p r o t e i n . I t was n o t
e s t a b l i s h e d , how ever, t h a t th e im m ediate p r e c u r s o r s o f a
g iv e n p r o t e i n m o lecu le a r e f r e e amino a c i d s . I t h as been
s u g g e s te d , f o r exam ple, t h a t a p r o t e i n may be pro d u ced by
th e assem bly o f p e p tid e fra g m e n ts o r by th e p a r t i a l d e
g r a d a t io n and re a ss e m b ly o f a n o th e r p r o t e i n m o le c u le .
Monod, e t a l . (9 ) and Spiegelm an and cow orkers
(1 0 , 11) d e m o n stra te d t h a t i n m ic ro o rg a n ism s, in d u ced
enzymes a r e form ed e x c l u s i v e l y from f r e e amino a c i d s . In
one e x p e rim e n t, Rotman and Spiegelm an (11) made
E s c h e r ic h ia c o l i u n ifo rm ly r a d i o a c t i v e by grow ing them on
iL
C - s u c r o s e . A f te r t r a n s f e r to a n o n - r a d io a c tiv e medium,
b e t a - g a l a c t o s i d a s e s y n th e s is was in d u c e d . A f te r some
tim e , th e b e t a - g a l a c t o s i d a s e was i s o l a t e d , p u r i f i e d , and
i t s r a d i o a c t i v i t y d e te rm in e d . W ith in e x p e rim e n ta l e r r o r ,
th e enzyme was n o t r a d i o a c t i v e and t h e r e f o r e had n o t been
d e riv e d i n any p a r t from th e r a d i o a c t i v e p r o t e i n o f th e
E . c o l i .
Simpson and V e lic k (12) and Heimberg and V e lic k
(13) have m easured th e r e l a t i v e r a t e s o f i n v iv o in c o rp o
r a t i o n o f s e v e r a l l a b e le d amino a c id s i n th e p r o t e i n o f
r a b b i t m uscle and c o n clu d ed t h a t e ac h p r o t e i n m ust be
d e riv e d from f r e e amino a c i d s . Askonas e t a l . (l^t)
i s o l a t e d some t h i r t y p e p tid e s from a p a r t i a l h y d r o ly s a te
o f c a s e in and b e t a - l a c t o g l o b u l i n o b ta in e d from th e m ilk
5
l k lli
o f a g o a t 3 h o u rs a f t e r i n j e c t i o n w ith C - v a l i n e and C -
l y s i n e . These two amino a c id s were found to have a
c o n s ta n t s p e c i f i c a c t i v i t y i n e v e ry p e p t i d e , w hich would
be an u n l ik e l y c o in c id e n c e u n le s s e v e ry p e p tid e had b een
d e riv e d from th e same p o o l— th e f r e e amino a c i d .
L o t f i e l d and H a r r is (15) in d u ced th e s y n t h e s i s o f
f e r r i t i n i n v iv o i n r a t l i v e r w h ile m a in ta in in g a c o n s ta n t
l k
i n t r a c e l l u l a r s p e c i f i c a c t i v i t y o f C - l e u c i n e , i s o l e u c i n e ,
and v a l i n e . A f te r 1 to 3 d a y s , f e r r i t i n was i s o l a t e d and
had l e u c i n e , i s o l e u c i n e , and v a li n e w ith th e same r a d i o
a c t i v i t y a s th e i n t r a c e l l u l a r f r e e amino a c i d p o o l and
much h ig h e r r a d i o a c t i v i t y th a n o th e r l i v e r p r o t e i n s . The
f e r r i t i n , t h e r e f o r e , c o u ld n o t have b een d e riv e d from
p r o t e i n s p r e s e n t when th e ex p erim e n t b eg an .
S tu d ie s o f th e p ro c e s s o f a n tib o d y fo rm a tio n have
y ie ld e d f u r t h e r e v id e n ce t h a t amino a c i d s a re th e immedi
a te p r e c u r s o r s o f b i o l o g i c a l l y f u n c t i o n a l p r o t e i n . A
r e c i p i e n t r a b b i t w i l l s y n th e s iz e a n ti b o d i e s i f he r e c e i v e s
a t r a n s p l a n t o f m inced s p le e n from a donor r a b b i t i n whom
a n tib o d y fo rm a tio n has b een in d u c e d . T a l i a f e r r o and
Talmage (16) have shown t h a t i f 5-am ino a c i d s w ere g iv e n
to th e donor sometime b e fo re t r a n s p l a n t , th e a n tib o d y
produced by th e r e c i p i e n t r a b b i t was n o n r a d i o a c t i v e . I f
S^5-amino a c id s were g iv e n to th e r e c i p i e n t a f t e r th e
t r a n s f e r o f s p le e n , th e a n tib o d ie s p ro d u ced were
r a d i o a c t i v e . T h is e x p e rim e n t d e m o n stra te d n o t o n ly t h a t
a n tib o d y p r o t e i n s a r e d e r iv e d from f r e e amino a c i d s , b u t
a ls o t h a t p r o t e i n p r e c u r s o r s o f a n tib o d y a r e n o t b e in g
form ed d u rin g th e 3 day p e rio d b e fo r e a n tib o d y i s p ro d u c e d .
A n fin se n and h i s c o v o rk e rs (1 7 -2 1 ) have o b se rv e d
s e v e r a l c a s e s , i n v i t r o and i n v i v o , where v a r io u s amino
a c id s a p p e a r to have b een in c o r p o r a te d i n t o d i f f e r e n t
p a r t s o f a p r o t e i n m o lecu le a t d i f f e r e n t r a t e s . They
s u g g e s t t h a t p e p tid e u n i t s l a r g e r th a n f r e e amino a c id s
to o k p a r t i n some o f th e r e a c t i o n s le a d in g to th e a p p e a r
ance o f th e i s o l a t e d p r o t e i n s . In a r e c e n t rev ie w
a r t i c l e , L o f t f i e l d (22) s u g g e s ts t h a t th e low r a t e o f
i n c o r p o r a ti o n o b se rv e d i n th e s e e x p e rim e n ts i s e i t h e r due
to r e a c t i o n s su b se q u e n t to th e a c t u a l a lp h a - p e p tid e c h a in
fo rm a tio n ( i . e . , se co n d a ry b o n d in g s) o r due to a v e ry much
slowed-down s y n t h e s i s o f th e a lp h a - p e p tid e c h a in . From a
d e t a i l e d su rv e y o f o b s e r v a tio n s from many l a b o r a t o r i e s ,
L o f t f i e l d (22) c o n c lu d e s t h a t , i n g e n e r a l , p r o t e i n syn
t h e s i s p ro c e e d s from f r e e amino a c id s w ith o u t s i g n i f i c a n t
p a r t i c i p a t i o n o f p e p tid e s u b u n i t s .
7
S tu d ie s o f P r o t e i n S y n th e s is I n
C e ll- F r e e P r e p a r a tio n s
C h a r a c t e r i s t i c s o f c e l l - f r e e system s
As lo n g a s th e c e l l rem a in s i n t a c t , i t i s a b le to
produce o r r e t a i n many o f th e m a t e r i a l s needed f o r b i o
c h em ic al a c t i v i t y . I f th e c e l l s t r u c t u r e i s a d e q u a te ly
d i s r u p t e d and f r a c t i o n a t e d , th e need f o r many e s s e n t i a l
f a c t o r s may become a p p a r e n t. For t h i s r e a s o n , many
w o rk ers e x te n d e d t h e i r e f f o r t s to c e l l - f r e e system s i n
stu d y in g th e b i o s y n t h e s is o f p r o t e i n . S e v e ra l p ro m in en t
w o rk ers i n th e f i e l d have d is c u s s e d th e l i m i t a t i o n s o f
th e s e c e l l - f r e e system s i n a com prehensive manner (2 2 -2 1 *).
I t would be i d e a l to have a system w hich would b r in g a b o u t
a d i r e c t l y m e a su ra b le n e t in c r e a s e i n p r o t e i n . Most
c e l l - f r e e system s th u s f a r s t u d i e d , how ever, have f a i l e d
to show a n e t s y n th e s is and th e in c o r p o r a ti o n o f r a d i o
a c t i v e amino a c id s i n to p r o t e i n h as g e n e r a l l y been used as
a m easure o f s y n t h e s i s .
E nergy so u rc e f o r p e p tid e bond fo rm a tio n
I n 19^1 Lipmann (25 ) su g g e ste d t h a t p h o sp h a te-b o n d
e n erg y dro v e th e p ro c e s s o f p r o t e i n s y n t h e s i s . The
l k
a v a i l a b i l i t y o f carb o n i n th e p o stw a r y e a rs made i t
p o s s ib l e f o r b io c h e m is ts to d e v is e e x p e rim e n ta l t e s t s o f
t h i s s u g g e s tio n . In l i v e r s l i c e s , u sin g d i n i t r o p h e n o l ,
F r a n tz e t a l . (26) found a p a r a l l e l i s m b etw een th e
I n h i b i t i o n o f p h o s p h o ry la tio n and th e i n h i b i t i o n o f amino
a c id i n c o r p o r a ti o n i n t o p r o t e i n .
As w ith th e t i s s u e s l i c e s t u d i e s , th e f i r s t
hom ogenates d e m o n s tra te d a r e l a t i o n betw een o x id a tiv e
p h o s p h o ry la tio n and amino a c i d i n c o r p o r a t i o n . W innick
( 2 7 ) , P e te rs o n e t a l . (2 8 , 2 9 ), and K it and G reenberg (30)
found t h a t a d e n y lic a c i d d e r i v a t i v e s s tim u la te d in c o r p o r a
t i o n . The o b s e r v a tio n was made by S ie k e v itz (31) t h a t
m ost o f th e la b e le d amino a c id e n te r e d microsom e p r o t e i n
b u t t h a t th e m ito c h o n d ria were a n e c e s s a r y component o f
t h e i r i n c o r p o r a ti o n sy ste m . He o b se rv e d t h a t th e in c o rp o
r a t i o n r e q u i r e d th o s e s u b s ta n c e s known t o be n e c e s s a r y f o r
o x id a tiv e p h o s p h o ry la tio n and r e p o r t e d , a s d id a ls o
A l l f r e y e t a l . ( 3 2 ) , t h a t r ib o n u c le a s e i n h i b i t e d th e i n
c o r p o r a tio n r e a c t i o n .
At ab o u t t h i s tim e , B ucher (33) made p o s s ib le a
m ajor advance i n t h i s ty p e o f stu d y when she found t h a t
l k
C - a c e t a t e c o u ld be b u i l t i n t o c h o l e s t e r o l i n a c e l l - f r e e
l i v e r hom ogenate, p ro v id e d t h a t g e n tl e h o m o g en iza tio n
( l o o s e ly f i t t i n g p e s t l e ) was em ployed. T hrough th e use o f
t h i s new te c h n iq u e , Zam ecnik and K e lle r (3*0 found t h a t i t
was p o s s ib l e to d i s c a r d th e m ito c h o n d ria l f r a c t i o n o f th e
c e l l - f r e e homogenate and t o use i n s t e a d , hexose d ip h o s
p h a te , p h o s p h o c re a tin e , o r p h o sp h o e n o lp y ru v ate a s an
e n erg y s o u r c e . A t t h i s p o i n t th e I n c o r p o r a ti o n system
c o n s is te d o f fo u r m ajor com ponents: (1 ) th e m icrosom e
f r a c t i o n (1 0 5 ,0 0 0 x g p r e c i p i t a t e a f t e r p r i o r rem oval o f
c e l l membrances and m ito c h o n d r ia ) , (2 ) th e l a b e le d amino
a c i d , (3) th e e n erg y d o n o r, and (*+) th e s o lu b le f r a c t i o n
(105 ,000 x g s u p e r n a ta n t f r a c t i o n ) . Upon d i a l y s i s o f th e
s o lu b le f r a c t i o n , th e system was i n a c t i v e u n le s s a d e n o sin e
tr ip h o s p h a te (ATP) and a h ig h -e n e rg y p h o sp h a te compound
such a s p h o s p h o c re a tin e were a l s o ad d ed .
Since p r o t e i n s y n th e s is , a s m easured by th e
in c o r p o r a ti o n o f amino a c id s i n t o m icrosom al p a r t i c l e s ,
was c o n s id e re d as p ro c e e d in g from f r e e amino a c i d s , i t was
re a s o n a b le f o r w orkers to i n v e s t i g a t e th e r o l e o f th e
en erg y s o u rc e , ATP, i n a c t i v a t i n g e i t h e r th e m icrosom es
o r th e amino a c id s th e m s e lv e s .
A c tiv a te d I n te r m e d ia te s in
P r o t e i n S y n th e s is
Amino a c id a c t i v a t i o n
The d is c o v e ry o f amino a c id a c t i v a t i o n was a
s i g n i f i c a n t advance tow ard u n d e rs ta n d in g th e manner i n
w hich f r e e amino a c id s may be s t a r t e d on th e pathw ay to
becoming com plete and d i s t i n c t p r o t e i n s . H oagland (35)
found t h a t th e s o lu b le enzyme f r a c t i o n needed f o r amino
a c id in c o r p o r a ti o n i n t o microsome p r o t e i n c a ta ly z e d an
10
amino a c i d d ep en d en t I n c o r p o r a ti o n o f p y ro p h o sp h a te (PP^)
I n to ATP. The a d d i t i o n o f a com plete complement o f
L-amino a c id s enhanced th e r a t e o f exchange o f r a d i o a c t i v e
PP^ i n to ATP. T h is f i n d in g was s u g g e s tiv e o f a p r o c e s s i n
w hich th e ATP r e a c t s r e v e r s i b l y w ith an L-am ino a c i d to
y i e l d an a m in o a c y l-a d e n y la te and P P ^ C a r b o x y l- a c tiv a te d
amino a c id s were tr a p p e d a s L-amino hydroxam ic a c id s by
th e use o f h ig h c o n c e n tr a tio n s o f hydro xylam ine ( 3 6 ) . The
e x te n t o f fo rm a tio n o f hydroxam ic a c id was d ep en d en t on
th e number o f s e p a r a te amino a c id s ad d ed . The f a c t t h a t
a m in o a c y l-a d e n y la te compounds were f i r m l y bound to enzymes
was su g g e ste d by th e f in d in g t h a t a d e n o sin e m onophosphate
(AMP) f a i l e d to exchange w ith ATP i n th e system and t h a t
no s o lu b le p ro d u c t was form ed i n th e ab sen ce o f an a c c e p to r
f o r th e a c t i v a t e d amino a c i d .
A c tiv a tio n o f amino a c id by th e m echanism
d e p ic te d i n R e a c tio n 1 h as been found i n a v a r i e t y o f
t i s s u e s a s a r e s u l t o f s t u d i e s by s e v e r a l g ro u p s o f
w ork ers (3 7 -^ 2 ) .
Amino a c id + ATP + a c t i v a t i n g enzyme 1 ** ^
Q am inoacyl— AMP— enzyme]] + PPj,
T h is r e a c t i o n r e q u ir e d no n u c le o ti d e s o t h e r th a n ATP and
c o u ld use no o t h e r s . H oagland e t a l . (36) found t h a t m ost
o f th e amino a c i d - a c t i v a t i n g enzym ic f r a c t i o n p r e c i p i t a t e s
11
a t pH 5 .2 , and p roduced an enhancem ent o f enzym ic a c t i v i t y
by t h i s p ro c e d u re . T h is f r a c t i o n has b e en r e f e r r e d t o a s
th e "pH 5 enzym es" w hich a re used by many w o rk e rs i n th e
stu d y o f p r o t e i n b i o s y n t h e s is i n c e l l - f r e e sy ste m s.
I t h as become a p p a r e n t, a s a r e s u l t o f s t u d i e s
r e p o r te d h e re and from s e v e r a l o th e r l a b o r a t o r i e s , t h a t
a l l amino a c id s may be a c t i v a t e d by th e same mechanism
(U-3— M-8). H ig h ly p u r i f i e d enzym es, s p e c i f i c f o r th e
a c t i v a t i o n o f s in g le amino a c i d s , have been i s o l a t e d (3 8 ,
*+9-5*0) and enzyme f r a c t i o n a t i o n s t u d i e s i n d i c a t e t h a t
t h e r e a re s e p a r a te a c t i v a t i n g enzymes f o r e a c h amino a c i d .
Much work h as now f ir m ly e s t a b l i s h e d th e m echanism (*+(),
5 5 -5 7 ). F i n a l u n e q u iv o c a l e v id e n ce has r e c e n t l y been
p r e s e n te d by Klngdon e t a l . (58) who have a c t u a l l y
i s o l a t e d a tr y p to p h a n a d e n y la te from l a r g e q u a n t i t i e s o f
p u r i f i e d tr y p to p h a r f - a c tiv a tin g enzyme.
Amino a c i d - r i b o n u c l e i c a c id fo rm a tio n
The a c t i v a t i o n o f amino a c id s was s tu d ie d f o r
s e v e r a l y e a rs b e fo re th e p h y s i o l o g ic a l a c c e p to r was
i d e n t i f i e d a s r i b o n u c l e i c a c id (RNA) by H oagland and co
w o rk ers ( 5 9 ). To th e i n i t i a l s u r p r i s e o f many b io c h e m is ts
i n th e f i e l d , th e RNA found t o be th e n a t u r a l c e l l u l a r
a c c e p to r o f a c t i v a t e d amino a c id s was t h e s o l u b l e , n o n -
m icrosom al RNA o f th e c e l l (59* 6 0 ) . M icrosom al
r i b o n u c l e o p r o t e i n p a r t i c l e s , w hich a r e r i c h i n RNA and
known t o be th e i n i t i a l s i t e o f p e p tid e bond c o n d e n s a tio n ,
had b een c o n s id e re d by many a s t h e "m ost l i k e l y c a n d i
d a te s " f o r th e d i r e c t t r a n s f e r o f a c t i v a t e d amino a c i d s .
The s i g n i f i c a n c e o f th e new f i n d in g was f u r t h e r r e a l i z e d
when i t was found (1 ) t h a t t h i s e v e n t o c c u rr e d e a r l i e r ,
i n v i v o , th a n a p p e a ra n c e o f amino a c i d i n p r o t e i n , and (2)
t h a t th e s o lu b le amino acid-RNA was a b le t o t r a n s f e r i t s
bound amino a c id to p a r t i c l e p r o t e i n (6 C ).
As so o fte n " h a p p e n s when th e tim e i s r i p e ,
s e v e r a l w o rk e rs were d o in g in d e p e n d e n t e x p e rim e n ts w hich
p o in te d tow ard such a s o lu b le i n t e r m e d i a t e . I n d i r e c t
e v id e n c e t h a t amino a c i d s became p r o g r e s s i v e l y bound to
some n o n p a r t i c u l a t e c e l l u l a r com ponent b e f o r e t h e i r
ap p e a ra n c e i n p a r t i c l e p r o t e i n was o b ta in e d by H u l t i n and
Beskow ( 6 1 ) . U sing th e amino a c i d i n c o r p o r a t i n g system o f
Zam ecnik and K e lle r (3*0» th e y f i r s t o b se rv e d t h a t th e
a d d i t i o n o f h e x o k in a se p lu s g lu c o s e t o th e f u n c t i o n in g
i n c o r p o r a t i o n system d id n o t s to p i n c o r p o r a t i o n immedi
a t e l y a lth o u g h th e e n e rg y s o u r c e , ATP, i s i n s t a n t l y
consum ed. In th e same way, th e a d d i t i o n o f one th o u s a n d -
12
f o l d e x c e s s o f C - l e u c i n e a f t e r th e i n c o r p o r a t i o n o f
C ^ - l e u c i n e had begun d id n o t im m e d ia te ly s to p th e i n
c o r p o r a t io n . F i n a l l y th e y in c u b a te d th e s o lu b le enzyme
f r a c t i o n w ith C ^ - l e u c i n e and th e ATP g e n e r a t i n g sy ste m ,
13
added th e C ^ - l e u c l n e , and th e n added m icro some s . The
microsom e p r o t e i n became l a b e le d even th o u g h th e m ic ro -
tL .
somes were n o t added u n t i l th e C - l e u c in e had been
d i l u t e d . These o b s e r v a tio n s a re c o n s i s t e n t w ith th e con
c e p t o f a n e n e rg y c o u p led p r o d u c tio n o f an " a c t i v a t e d "
amino a c id w hich does n o t r a p i d l y e q u i l i b r a t e w ith th e
f r e e amino a c i d p o o l b u t w hich does r e a c t q u ic k ly and
e x e r g o n i c a ll y w ith m icrosom es to form p r o t e i n .
H o lle y (62) showed t h a t th e r a t l i v e r pH 5 enzymes
c a ta ly z e d an a la n in e - d e p e n d e n t, r ib o n u c le a s e s e n s i t i v e
it.
i n c o r p o r a ti o n o f C - l a b e l e d AMP i n t o ATP, s u g g e s tin g th e
r e v e r s i b l e f o rm a tio n o f an RN A -alanine compound. Ogata
e t a l . (63) o b se rv ed an i n h i b i t o r y e f f e c t o f r ib o n u c le a s e
upon amino a c id a c t i v a t i o n i n r a b b i t t i s s u e s w hich l e d to
d i r e c t o b s e r v a tio n s on t r a n s f e r o f amino a c id s to a
s o lu b le RNA. A s h o r t tim e l a t e r , t h e r e were r e p o r t s con
c e rn in g th e fo rm a tio n o f amino acid-RNA compounds (R eac
t i o n 2) from s e v e r a l l a b o r a t o r i e s , i n one o f w hich th e
e a r l y s ta g e s o f r e s e a r c h r e p o r te d i n t h i s d i s s e r t a t i o n
were ta k in g p l a c e .
^Aminoacyl— AMP— enzyme^J + RNA ---^ ^
Amino a c i d —RNA + AMP + enzyme
Schweet e t a l . (6M-), Berg and Ofengand (65)» and W ebster
(66) found e v id e n c e f o r amino acid-RNA fo rm a tio n i n g u in e a
p i g , b a c t e r i a l , and p l a n t t i s s u e s , r e s p e c t i v e l y . A
s o lu b le c o f a c t o r w hich s tim u la te d i n c o r p o r a ti o n o f amino
a c id s i n t o p r o t e i n i n a c e l l - f r e e p a n c re a s system was
d is c o v e re d by W eiss e t a l . (67) and was s u b s e q u e n tly
shown to be a p o ly n u c le o tid e .
The BNA w hich p a r t i c i p a t e s i n R e a c tio n 2 h a s th u s
su d d en ly ta k e n th e l i m e l i g h t i n th e stu d y o f p r o t e i n
s y n t h e s i s . The n a tu r e o f t h i s RNA and th e p r o c e s s by
w hich amino a c id s become lin k e d to i t were th e o b j e c t s o f
th e s t u d i e s p r e s e n te d h e r e . The s i g n i f ic a n c e o f th e r e a c
t i o n seems to be i n u n e q u iv o c a lly r e l a t i n g th e m etab o lism
o f th e b u ild in g b lo c k s o f p r o t e i n w ith th e su b s ta n c e
known f o r many y e a rs to be i n t i m a t e l y in v o lv e d i n th e
s y n th e s is o f p r o t e i n .
II. STATEMENT OF THE PROBLEM
AND PLAN OF ATTACK
The p ro c e s s by w hich amino a c i d s become l in k e d to
RNA and th e n a tu r e o f th e RNA in v o lv e d were o b j e c t s o f th e
s t u d i e s p r e s e n te d h e r e .
The s o lu b le enzyme f r a c t i o n from guinea p ig l i v e r
was ch o sen f o r s tu d y s in c e p r e lim in a r y i n v e s t i g a t i o n had
shown t h a t th e pH 5 enzymes p re p a re d from t h i s so u rce were
v e ry a c t i v e i n c a ta ly z in g amino a c id a c t i v a t i o n and i n
c o r p o r a tio n i n t o RNA. The r e s e a r c h p la n in v o lv e d a s tu d y
o f amino a c id a c t i v a t i o n i n an a tte m p t to d e te rm in e
w h eth er a l l o f th e common amino a c id s n o rm a lly found i n
p r o t e i n s may be a c t i v a t e d by th e same m echanism .
I n a d d i t i o n to t h i s , i t was th e o b j e c t o f th e
s t u d i e s to d e s ig n m ethods f o r s e p a r a t io n o f RNA from
a c t i v a t i n g enzymes and th e f u r t h e r f r a c t i o n a t i o n o f th e s e
enzymes so t h a t a more d e t a i l e d i n v e s t i g a t i o n o f amino
acid-RNA fo rm a tio n c o u ld be made. Through th e use o f
enzyme f r a c t i o n s w hich would c a ta ly z e th e a c t i v a t i o n o f
o n ly one o r a few amino a c i d s , i t was th o u g h t p o s s ib l e to
stu d y th e d i r e c t r e l a t i o n s h i p betw een th e p r o p e r t i e s o f
a c t i v a t i n g enzymes and th e r e a c t i o n o f amino a c i d w ith RNA.
15
16
S in ce i t had b e en I n d i c a te d t h a t amino acid-RNA
fo rm a tio n was an in te r m e d ia te r e a c t i o n i n p r o t e i n sy n th e sis
( 6 0 ) , th e u ltim a te g o a l o f th e r e s e a r c h s t u d i e s , i f tim e
were a v a i l a b l e , would be t o p ro v id e a d d i t i o n a l e v id e n c e
c o n c e rn in g th e s t a t u s o f t h i s r e a c t i o n a s an in te r m e d ia te
s t e p . T h is problem would a ls o in v o lv e a stu d y o f th e
n a tu r e o f th e " t r a n s f e r RNA"— a term w hich d e n o te s i t s
f u n c t i o n i n t r a n s f e r r i n g a c t i v a t e d amino a c i d s to th e
m icrosom al p a r t i c l e s , th e s i t e o f p r o t e i n s y n t h e s i s .
III. MATERIALS AND METHODS
M a t e r i a l s
A nim als
G uinea p ig s and r a b b i t s w ere o b ta in e d from th e
V a lle y L a b o ra to ry Supply Company, La P u e n te , C a l i f o r n i a .
G uinea p i g s (1 8 -2 0 o u n c e s) were used f o r th e l i v e r enzyme
p r e p a r a t i o n s . F iv e t o s i x pound New Z e a la n d w h ite r a b b i t s
were used i n th e s t u d i e s o f hem oglobin s y n t h e s i s .
C hem icals
N u c le o s id e s , n u c l e o t i d e s , n u c le o s id e d ip h o s p h a te s
and t r i p h o s p h a t e s were o b ta in e d from P a b s t L a b o r a t o r i e s .
G lu ta th io n e (GSH) was o b ta in e d from Schwarz L a b o r a t o r i e s .
P ro tam in e s u l f a t e and C -am ino a c i d s w ere p r o d u c ts o f th e
N u t r i t i o n a l B io c h e m ic a ls C o r p o r a tio n . C r e a tin e p h o sp h a te
was o b ta in e d from th e C a l i f o r n i a C o r p o r a tio n f o r B io
c h e m ic a l R e s e a rc h . T r i s (hydroxym ethyl)am inom ethane and
p - c h lo r o m e r c u rib e n z o ic a c i d were o b ta in e d from Sigma
C hem ical Company. O th er common c h e m ic a ls were th o s e
o b ta in e d from s ta n d a r d com m ercial s o u r c e s .
17
18
I s o t o p i c compounds
ATP-8-C-*-1 + (218 c .p .m . p e r mumole) was o b ta in e d
from Schwarz L a b o r a t o r i e s . U n ifo rm ly l a b e l e d L - le u c i n e -
C ^ , L - ls o le u c i n e - C ^ +, L -th re o n ln e -C ^ 1 *, L - ly s in e - C ^ " ,
ILl i L. i L
L - ty r o s in e - C , L - v a l i n e - C , L -a r g in in e -C , L - a l a n i n e -
C^*, L -g lu ta m ic a c id - C ^ * , L - p h e n y la la n ln e -C ^ * , g l y c i n e -
C ^ , and D L -try p to p h a n ( i n d o l y l a la n in e -3 -C -1 -lf) were
o b ta in e d from N u c le a r-C h ic a g o C o r p o r a tio n . D L -le u c in e -1 -
was o b ta in e d from th e C a l i f o r n i a C o r p o ra tio n f o r
B io c h e m ic a l R e s e a rc h .
R a d io a c tiv e p o ta s s iu m p y ro p h o sp h a te was p re p a r e d
by p y r o l y s i s (6 8) o f th e d ip o ta s s iu m s a l t o f P ^ - l a b e l e d
o rth o p h o s p h a te , o b ta in e d from Oak R idge N a tio n a l L ab o ra
t o r i e s . The P P ^ 2 used c o n ta in e d l e s s th a n % o f i n
o r g a n ic p h o sp h a te and no p o ly p h o sp h a te d e t e c t a b l e by
chro m atography ( 6 9 ) .
O ther m a t e r i a l s
C r y s t a l l i n e r ib o n u c le a s e was p u rc h a se d from
W o rth in g to n B io c h e m ic a l S a le s Company. C alcium p h o sp h a te
g e l , p re p a re d by th e m ethod o f S in g e r and K earney ( 7 0 ) ,
was aged s e v e r a l m onths b e fo re use and c o n ta in e d 30 mg o f
dry m a t e r i a l p e r m l.
C r e a tin e k in a s e was p re p a r e d and p u r i f i e d th ro u g h
th e a lc o h o l f r a c t i o n a t i o n by th e m ethod o f Kuby e t a l .
( 7 1 ) .
19
C r y s t a l l i n e y e a s t p y ro p h o sp h a ta se was k in d ly
d o n a te d by D r. M. K u n itz , R o c k e f e lle r I n s t i t u t e f o r
M edical R e se a rc h , New York C ity , New Y ork.
E . c o l l RNA was k in d ly p ro v id e d by D r. P a u l R e rg ,
D epartm ent o f M ic ro b io lo g y , W ashington U n i v e r s i ty , S t .
L o u is , M is s o u ri.
Methods
A ssay p ro c e d u re s
P yrophosph ate exchange a s s a y . — The r e a c t i o n
m ix tu re f o r th e p y ro p h o sp h ate exchange a s s a y has been
d e s c rib e d by Schweet and A lle n (5 0 ). The o n ly change in
th e a s s a y p ro ce d u re was th e use o f T r is b u f f e r , pH 8 .5
(pH a d ju s te d a t 2 5 ° ) , i n s t e a d o f g ly c in e b u f f e r . The
r a d i o a c t i v i t y i n ATP was d e te rm in e d by a m o d if ic a tio n (*+9)
o f th e method o f Crane and Lipmann ( 7 2 ) . The p e rc e n ta g e
o f exchange and c o n v e rsio n to umoles was c a l c u l a t e d a s
d e s c r ib e d by Davie e t a l . (5 l)»
A u n i t o f enzyme a c t i v i t y i s d e fin e d a s th e amount
o f enzyme t h a t c a ta ly z e s th e in c o r p o r a ti o n o f 1 .0 jomole
o f PP^ i n t o ATP i n 10 m in u te s i n th e s ta n d a rd exchange
a s s a y . S p e c if ic a c t i v i t y i s g iv e n i n u n i t s p e r mg o f
p r o t e i n .
Amino a c id i n c o r p o r a ti o n i n t o r i b o n u c l e i c a c i d . —
The r e a c t i o n m ix tu re f o r amino a c id i n c o r p o r a ti o n i n t o RNA
c o n ta in e d a p p ro x im a te ly 0 .2 t o 1 .0 u n i t o f enzyme; 10
pm oles o f d ip o ta s s iu m ATP, a d ju s te d to pH 7 . 5 w ith
p o ta ssiu m h y d ro x id e ; 10 p i o l e s o f magnesium c h l o r i d e ; 100
lU-
p n o le s o f T r i s b u f f e r , pH 7 .5 ; 0 .0 2 5 pm oles o f a C -am ino
a c i d ; and w a te r i n a f i n a l volume o f 1 .0 m l. RNA p re p a re d
by a p h e n o l m ethod (73) was added to th e r e a c t i o n m ix tu re
when n e c e s s a r y . When sm a ll am ounts o f p u r i f i e d a c t i v a t i n g
enzymes were u se d , 1 .0 mg o f serum alb u m in and 10 jimoles
o f g l u ta t h io n e were a ls o ad d ed .
A f te r in c u b a tio n f o r 10 m in u te s a t 37° i n a
D ubnoff m e ta b o lic s h a k e r , th e r e a c t i o n m ix tu re was q u ic k ly
c h i l l e d , c a s e in was added to g iv e 15 mg o f p r o t e i n , and
th e r i b o n u c l e o p r o t e i n s p r e c i p i t a t e d w ith 8 volum es o f
3 .5 $ t r i c h l o r o a c e t i c a c i d . The p r e c i p i t a t e was washed by
a m o d if ic a tio n o f th e m ethod o f H oagland e t a l . ( 5 9 ).
A f te r w ashing 3 tim e s w ith c o ld 0 .2 M p e r c h l o r i c a c i d ,
^ ml o f 5 s i e th a n o l: 0 .2 M p e r c h l o r i c a c id was ad d ed .
A f te r th o ro u g h m ixing i n th e e t h a n o l - p e r c h l o r i c a c i d ,
6 ml o f e t h e r was added to r e p r e c i p i t a t e p r o t e i n and th e
m ix tu re was a llo w e d to s ta n d f o r 20 m in u te s . The p r e
c i p i t a t e was th e n washed once w ith 3 s i e t h a n o l : e t h e r f o r
10 m in u te s a t 6 0 ° , and f i n a l l y tw ic e w ith e t h e r . The d ry
sam ples were d i s t r i b u t e d w ith benzene i n a co pper
p l a n c h e t , d r i e d , and th e r a d i o a c t i v i t y d e te rm in e d w ith a
N u c lea r "M ic ro m il1 1 window g a s - flo w c o u n te r . When
21
1^
a v a i l a b l e , th e C -am ino a c id s were used a t a s p e c i f i c
a c t i v i t y o f 7 fic u rie s p e r fimole, e q u iv a le n t to 2 .2 x 10^
c o u n ts p e r m inute ( c .p .m .) on o u r c o u n te r s . I f amino
a c id s w ith low er s p e c i f i c a c t i v i t i e s were u se d , r e s u l t s
were c o r r e c t e d to c o rre sp o n d w ith t h i s v a lu e .
ill
ATP i n c o r p o r a ti o n i n t o r i b o n u c l e i c a c i d . — CA -ATP
i n c o r p o r a ti o n i n to RNA was d e te rm in e d w ith th e same
in c u b a tio n m ix tu re used f o r amino a c id i n c o r p o r a ti o n i n to
12 1 * +
RNA , e x c e p t t h a t C -am ino a c id s and 1 .0 ^unole o f C -ATP
were u se d . The r a d i o a c t i v i t y i n th e RNA was d e te rm in e d a s
d e s c r ib e d f o r amino a c id i n c o r p o r a ti o n .
Amino a c id i n c o r p o r a ti o n i n t o p r o t e i n . — The
com plete r e a c t i o n m ix tu re c o n ta in e d 0 .5 ml o f rib o so m es
to 8 mg d ry w e ig h t) p re p a re d from r a b b i t r e t i c u l o c y t e s ;
1 to 2 mg o f enzyme (Ammonium s u l f a t e f r a c t i o n , to 70%
s a t u r a t i o n (AS ^ 0 - 7 0 ) ) ; 50 to 200 fig o f RNA p re p a re d by a
p h e n o l m ethod; 120 fig o f c r e a t i n e k i n a s e ; 1 .0 fimole o f
ATP, pH 7 -5 ; 50 fimoles o f T r i s c h lo r id e b u f f e r , pH 7*5;
10 fimoles o f c r e a t i n e p h o s p h a te , a d ju s te d to pH 7 .0 w ith
h y d r o c h lo r ic a c i d ; 20 fimoles o f GSH; 100 fimoles o f p o t a s
sium c h l o r i d e ; 5 fimoles o f magnesium c h l o r i d e ; 0 .2 5 fimole
o f g u a n o sin e tr ip h o s p h a te (GTP); 0 .1 fimole o f a C -^-am ino
a c i d , d i l u t e d to a s p e c i f i c a c t i v i t y o f 3000 c .p .m . p e r
mfimole; and th e com plete amino a c i d m ix tu r e , i n a f i n a l
volume o f 1.*+ m l. The com plete amino a c i d m ix tu re
22
c o n ta in e d th e e ig h t e e n amino a c i d s i n th e p r o p o r t i o n s , b u t
a t o n e - s i x t h th e f i n a l c o n c e n tr a ti o n d e s c r i b e d by B o rso ok
and co w o rk ers (7*0» The C - l a b e l e d amino a c i d und er
s tu d y was o m itte d from th e m ix tu re o f u n la b e le d amino
lU. I 1 * -
a c i d s . W ith D L - le u c in e - l- C , 0 .2 pmole o f th e C -am ino
a c i d was u s e d .
A f te r in c u b a ti o n f o r M -5 m in u te s a t 37° i n a
D ubnoff m e ta b o lic s h a k e r , th e p r o t e i n s were p r e c i p i t a t e d
and washed once w ith 5# t r i c h l o r o a c e t i c a c i d , d i s s o l v e d
i n 0 .5 ml o f IK sodium h y d ro x id e , r e p r e c i p i t a t e d and
w ashed once w ith 5# t r i c h l o r o a c e t i c a c i d . The p r e c i p i t a t e
was th e n s t i r r e d f o r 5 t o 10 m in u te s i n 3 ml o f 95#
e th a n o l ( h e a tin g a t 60° f o r 10 m in u te s a t t h i s s te p
p ro d u ced e q u iv a le n t r e s u l t s ) . Two volum es o f e t h e r w ere
added t o in s u r e p r e c i p i t a t i o n o f p r o t e i n and th e m ix tu re
was a llo w e d to s ta n d f o r 20 m in u te s . The p r e c i p i t a t e was
f i n a l l y w ashed tw ic e w ith e t h e r , d r i e d , p l a t e d , and th e
r a d i o a c t i v i t y d e te rm in e d , a s d e s c r ib e d b e f o r e . The d r i e d
p r o t e i n s were w eighed and a l l r e s u l t s c o r r e c t e d f o r s e l f
a b s o r p t i o n . The r e s u l t s a r e g iv e n a s c .p .m . p e r mg o f
m icro so m es, b a se d on th e a c t u a l d ry w e ig h t o f a sam ple o f
m icrosom es washed a s d e s c r ib e d a b o v e. T h is p r e c i p i t a t e
was found by r i b o s e a n a l y s i s to c o n ta in 30 to Ho p e r c e n t
RNA.
v
A n a l y ti c a l m ethods
P r o t e in was d e te rm in e d b y l i g h t a b s o r p t io n ( 7 5 )
e x c e p t i n f r a c t i o n s w ith a h ig h RNA c o n te n t where th e
method o f Lowry e t a l . (76) was u se d .
The RNA c o n te n t o f s o l u t io n s c o n ta in in g p r o t e i n
was d e te rm in e d by an o r c i n o l m ethod (7 7 )• W ith i s o l a t e d
RNA, th e o p t i c a l d e n s i t y a t 260 mfi i n 0 .0 2 M T r i s b u f f e r ,
1%
pH 7 . 5 , w i t h t h e v a l u e cm = 230, w a s u s e d f o r q u a n t i t a
t i v e e s t i m a t i o n .
P hosph ate was d e te rm in e d by th e method o f D ryer
e t a l . ( 7 8 ) .
Enzyme p r e p a r a t i o n
P r e p a r a tio n o f pH 5 enzym es. — G uinea p ig s were
f a s t e d f o r 16 to 18 h o u r s , k i l l e d , and th e l i v e r s q u ic k ly
e x c is e d and p la c e d in an ic e b a th . A p p ro x im ately 70 g o f
l i v e r were m inced w ith s c i s s o r s and th e n hom ogenized i n
15 t o 20 gram p o r t io n s i n 2 volum es (1^-0 m3) o f medium A
(79) f o r 15 to 20 seconds w ith a P o tte r-E lv e h je m
h o m og enizer. The te m p e ra tu re was k e p t a t 0 t o b ° a t a l l
tim e s d u rin g th e p r e p a r a t i o n and f r a c t i o n a t i o n o f th e
enzymes u n le s s o th e rw is e n o te d . The homogenate ( a p p r o x i
m a te ly 200 ml) was c e n tr if u g e d a t 1 5 ,0 0 0 x g f o r 10 m in
u te s to remove c e l l d e b r i s . The s u p e r n a ta n t (l*+0 m l) was
f i l t e r e d th ro u g h g l a s s wool and c e n tr if u g e d a t 10 5,000 x
2b
g f o r 1 h o u r . The c l e a r s u p e r n a ta n t f l u i d (110 m l)
c o n ta in in g th e s o lu b le c e l l f r a c t i o n was p i p e t t e d o u t .
The s o l u t i o n was c a r e f u l l y a d j u s t e d to pH 5*15 by th e
a d d i t i o n o f 1 N a c e t i c a c i d w ith c o n s ta n t s t i r r i n g . The
p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and r e s u s
pended i n a b o u t 30 ml o f 0 .1 M T r i s b u f f e r , pH 7 . 5 , w ith
v e ry b r i e f h o m o g e n iz a tio n . The s o l u t i o n was r e p r e c i p i t a t e d
a t pH 5 .1 5 and th e p r e c i p i t a t e ta k e n up i n s u f f i c i e n t 0 .1
M T r i s , pH 7 . 5 , t o g iv e a s o l u t i o n c o n ta in in g 12 mg o f
p r o t e i n p e r ml (pH 5 enzym es, $b m l) . The pH 5 enzymes
were Im m e d ia tely f r a c t i o n a t e d a s d e s c r ib e d below o r w ere
s t o r e d a t -1 5 ° i n th e p re s e n c e o f 0 .0 5 M GSH and 0 .0 0 5 M
e th y le n e d ia m in e t e t r a a c e t i c a c id (EDTA).
F r a c t i o n a t i o n o f p H 5 enzym es. — The f r a c t i o n a t i o n
scheme ( F ig . 1) u t i l i z e s th e a f f i n i t y o f b o th tra n sfe r-R N A
(73) and a c t i v a t i n g enzymes f o r c a lc iu m p h o sp h a te g e l .
S ince t r a n s f e r RNA and two o f th e a c t i v a t i n g enzymes
(Ammonium s u l f a t e f r a c t i o n 1 (A S -1)) a r e s o lu b le i n 2 M
p h o sp h a te b u f f e r , th e s e a r e s e p a r a te d from o t h e r a c t i v a t
in g enzymes by th e e l u t i o n sequence shown.
E ig h te e n m i l l i t e r s (1 /3 volum e) o f aged c a lc iu m
p h o sp h a te g e l (70) w ith a d ry w e ig h t o f 30 mg p e r ml were
added to th e pH 5 enzyme s o l u t i o n . The m ix tu re was
s t i r r e d g e n t l y f o r b $ m in u te s , and th e n c e n t r if u g e d a t
3500 x g f o r 10 m in u te s . The s u p e r n a ta n t s o l u t i o n ( g e l
25
pH 5 ENZYMES
GEL
ABSORPTION
SUPER (P/SCARP)
ELUATE 1
GEL PPT.
2 M
PHOSPHATE
ELUTION
-► GEL PPT.
PROTAMINE
SUPER 1 PPT.
SUPER
(P/SCARP)
9 0 %
AS
0.2 M PHOSPHATE
ELUTION
ELUATE 2 GEL PPT. (P/SC AR D)
PHENOL PROTAMINE
EXTRACTION
TRANSFER-RNA
PPT. (A H )
-► m .m e arp)
SUPER 2
30% A S
su
40%AS
SU
60%AS
80%AS
-►PPT(P /SCA RP )
PER
^ P P IC A S -2 )
PER
-► P P K A S -3 )
SUPER
-► PPT.(AS-4)
SUPER ( P /S C A R P )
F ig u re 1 . —F r a c t i o n a t i o n scheme f o r pH 5 enzym es.
D e t a i l s o f th e f r a c t i o n a t i o n a re g iv e n i n th e t e x t .
Ammonium s u l f a t e f r a c t i o n s a re a b b r e v ia te d a s AS.
s u p e r) was u s u a l ly d i s c a r d e d . The p r e c i p i t a t e d g e l was
th e n m ixed w ith 35 ml o f 2 M p o ta s s iu m p h o sp h a te b u f f e r )
pH 8 .1 ( 2 /3 v o lu m e ). The m ix tu re was hom ogenized b r i e f l y ,
s t i r r e d g e n tl y o v e r a p e r io d o f *+5 m in u te s , and c e n tr if u g e d
f o r 10 m in u te s a t 1 5 ,0 0 0 x g . The s u p e r n a ta n t s o l u t i o n
was d l a l y z e d w ith s t i r r i n g f o r 18 h o u rs a g a i n s t two
changes o f 2 l i t e r s o f 0 .0 2 M T r i s b u f f e r , pH 7 .5 (E lu a te
I , 55 m l) . The p r e c i p i t a t e d g e l was im m e d ia te ly m ixed
w ith 18 ml o f 0 .2 M p o ta s s iu m p h o sp h a te b u f f e r , pH 8 .1
(1 /3 T v o lu m e ). The s l u r r y was s t i r r e d and c e n t r i f u g e d a s
b e f o r e , and th e p r e c i p i t a t e d g e l d i s c a r d e d . The s u p e r
n a t a n t can be s t o r e d a t -1 5 ° (w ith 0 .0 5 M GSH p lu s 0 .0 0 5
EDTA) b e f o r e f u r t h e r f r a c t i o n a t i o n ( E lu a te 2 , 18 m l) , b u t
was u s u a l l y f r a c t i o n a t e d a t o n c e . F o r a s s a y , a l i q u o t s
were d l a ly z e d a g a i n s t 2 l i t e r s o f 0 .0 2 M T r i s b u f f e r , pH
7 . 5 , c o n ta in in g 1 x 10“ 3 M GSH and 1 x 1 0 M EDTA ( T r i s -
GSH-EDTA b u f f e r ) .
A f te r d i a l y s i s , E lu a te 1 was a d ju s te d to a T r i s
b u f f e r c o n c e n tr a ti o n o f 0 .1 2 M by th e a d d i t i o n o f 2 .8 0 ml
o f 2 M T r i s b u f f e r , pH 7 .5 * The 260 mp a b s o r p t io n was
d e te rm in e d and p ro ta m in e s u l f a t e s o l u t i o n (10 m g/m l) was
th e n added i n sm a ll am o u n ts. A liq u o ts w ere c e n t r if u g e d
and th e 260 mp a b s o r p t io n o f th e s u p e r n a ta n t d e te rm in e d .
A d d itio n o f p ro ta m in e s u l f a t e was c o n tin u e d u n t i l 85 to
90% o f th e 260 mp a b s o rb in g m a t e r i a l had p r e c i p i t a t e d .
T h is u s u a l l y r e q u i r e d a b o u t 1 . 5 mg o f p ro ta m in e s u l f a t e
p e r mg o f RNA. The m ix tu re was c e n t r i f u g e d a f t e r 20
m in u te s and th e p r e c i p i t a t e sav ed f o r p r e p a r a t i o n o f
t r a n s f e r RNA. The p r o t e i n o f th e s u p e r n a ta n t s o l u t i o n
(S u p er 1 , 60 m l) was th e n p r e c i p i t a t e d by th e a d d i t i o n o f
ammonium s u l f a t e t o a t t a i n 90 p e r c e n t s a t u r a t i o n ( 3 6 .2 g ) .
A f te r s ta n d in g f o r 2 h o u rs w ith o c c a s i o n a l s t i r r i n g , th e
p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n . The
s u p e r n a ta n t s o l u t i o n was d i s c a r d e d . The p r e c i p i t a t e was
d is s o lv e d i n 5 ml o f 0 .1 M T r i s b u f f e r , pH 7 - 5 * and
d la ly z e d o v e r n ig h t a g a i n s t 0 .0 2 M T r i s , pH 7 . 5 ( F r a c t i o n
A S-1, 7 .2 m l).
E lu a te 2 was f r e e d o f r e s i d u a l t r a n s f e r RNA by th e
a d d i t i o n o f p ro ta m in e s u l f a t e s o l u t i o n to a f i n a l co n cen
t r a t i o n o f 0 .3 5 mg o f p ro ta m in e s u l f a t e p e r m l. The
p r e c i p i t a t e d protam ine-RN A was d is c a r d e d and th e s u p e r
n a t a n t s o l u t i o n was d i a ly z e d o v e r n ig h t a g a i n s t th e T r i s -
GSH-EDTA b u f f e r d e s c r ib e d a b o v e . T r i s b u f f e r , pH 7 .5 » was
th e n added to th e d i a l y z e d enzyme so t h a t th e f i n a l b u f f e r
c o n c e n tr a ti o n was 0 .1 2 M and th e p r o t e i n c o n c e n tr a ti o n
5 mg p e r ml (S u p e r 2 , 22 m l) . Powdered ammonium s u l f a t e
was added to a t t a i n 30# s a t u r a t i o n ( 3 .6 and th e
m ix tu re was c e n t r i f u g e d a f t e r 30 m in u te s . The p r e c i p i t a t e
was d i s c a r d e d , and ammonium s u l f a t e was added t o b r in g th e
s u p e r n a ta n t t o k-0% s a t u r a t i o n (1 .3 7 g ) . The m ix tu re was
28
c e n t r i f u g e d a f t e r 30 m in u te s and th e p r e c i p i t a t e d p r o t e i n
was d i s s o l v e d i n 5 ml o f 0 .1 M T r i s b u f f e r , pH 7 .5 * and
d i a ly z e d a g a i n s t th e Tris-GSH-EDTA b u f f e r ( F r a c t i o n A S -2 ).
Ammonium s u l f a t e was t h e n added t o th e s u p e r n a ta n t to
a t t a i n (>0% s a t u r a t i o n (2*97 g ) . A f te r 30 m in u te s , th e
p r e c i p i t a t e d p r o t e i n was c o l l e c t e d by c e n t r i f u g a t i o n , d i s
so lv e d i n 5 ml o f 0 .1 M T r i s b u f f e r , pH 7»5» and d i a ly z e d
a g a i n s t Tris-GSH-EDTA b u f f e r ( F r a c t i o n A S -3 ). The s u p e r
n a t a n t was th e n ta k e n t o 80# s a t u r a t i o n by th e f u r t h e r
a d d i t i o n o f s o l i d ammonium s u l f a t e (3*^-2 g ) . The m ix tu re
was a g a in c e n t r if u g e d a f t e r 30 m in u te s , th e s u p e r n a ta n t
d i s c a r d e d , and th e p r e c i p i t a t e d p r o t e i n d i s s o l v e d i n 2 t o
3 ml o f 0 .1 M T r i s b u f f e r , pH 7.5» and d i a l y z e d a g a i n s t
Tris-GSH-EDTA b u f f e r ( F r a c t i o n AS-1 * ).
Ribosome p r e p a r a t i o n from r a b b i t r e t i c u l o c y t e s . —
R a b b it r e t i c u l o c y t e s w ere p r e p a re d and washed by a
m o d if i c a t io n o f th e m ethod o f B orsook e t a l . ( 8 0 ) .
R a b b its w ere i n j e c t e d s u b c u ta n e o u s ly f o r 5 d ay s w ith 2.5%
p h e n y lh y d r a z in e , n e u t r a l i z e d to pH 7 .0 w ith sodium
h y d ro x id e . The dosage f o r a 5 pound r a b b i t was 1 .0 cc p e r
d a y . No i n j e c t i o n was made on th e s i x t h day a f t e r i n i t i a
t i o n o f th e i n j e c t i o n s , and th e r a b b i t s were b le d by h e a r t
p u n c tu re under e t h e r a n e s t h e s i a on th e s e v e n th d a y . The
h e p a r i n iz e d b lo o d was c e n t r i f u g e d a t 2500 x g and th e
plasm a d i s c a r d e d . The c e l l s were w ashed w ith an i s o t o n i c
s o l u t i o n c o n ta in in g 0 .1 3 M sodium c h l o r i d e , 0 .0 0 5 M
p o ta s s iu m c h l o r i d e , and 0 .0 0 7 5 M m agnesium c h l o r i d e . A ll
s t e p s w ere c a r r i e d o u t a t 4-° u n le s s o th e r w is e i n d i c a t e d .
A f te r f i l t e r i n g th ro u g h g a u z e , th e c e l l s were a g a in
c e n t r i f u g e d a t 2500 x g f o r 10 m in u te s . The p ack ed c e l l s
( u s u a l ly 50 ml from t h r e e r a b b i t s ) w ere l y s e d by th e
a d d i t i o n o f 4 - volum es o f 0 .0 0 2 5 M magnesium c h lo r i d e (200
m l) , and th e m ix tu re was s t i r r e d g e n t l y f o r 10 m in u te s .
One volume o f 1 .5 M su c ro s e (50 m l) c o n ta in in g 0 .1 5 M
p o ta s siu m c h lo r id e was added s lo w ly , and th e m ix tu re
c e n t r i f u g e d a t 1 5 ,0 0 0 x g f o r 10 m in u te s . The p r e c i p i t a t e ,
c o n ta in in g c e l l m em branes, m ito c h o n d r ia , and unbroken
c e l l s , was d i s c a r d e d . The s o l u t i o n was th e n c e n t r if u g e d
f o r 1£ h o u rs a t 7 8 ,4 0 0 x g , o r f o r 1 h o u r a t 10 5 ,5 0 0 x g ,
y i e l d i n g a p r e c i p i t a t e and s u p e r n a ta n t (S u p er 3 , 250 m l) .
The p r e c i p i t a t e (rib o so m e s) was w ashed by su sp en d in g i t i n
a p p ro x im a te ly 200 ml o f Medium B (0 .2 5 M s u c r o s e , 0 .0 1 7 5 M
p o ta s siu m b i c a r b o n a t e , 0 .0 0 2 M magnesium c h l o r i d e ) and was
c e n t r i f u g e d a g a in f o r l-J- h o u rs a t 78,4-00 x g . The s u p e r
n a t a n t (S u p er 3 from th e h ig h speed c e n t r i f u g a t i o n was
used f o r th e p r e p a r a t i o n o f r e t i c u l o c y t e s enzymes and RNA
d e s c r ib e d i n th e f o llo w in g s e c t i o n s . The f i n a l w ashed
ribosom e p e l l e t was suspended i n 0 .2 5 M s u c r o s e , y i e l d i n g
a p a le - y e llo w o p a le s c e n t s o l u t i o n .
30
P r e p a r a t i o n o f enzyme f r a c t i o n from r a b b i t
r e t i c u l o c y t e s . — A f te r rem oval o f r e t i c u l o c y t e rib o so m e s by
h ig h sp eed c e n t r i f u g a t i o n , th e s u p e r n a ta n t s o l u t i o n (S u p er
3 , 250 m l) , was r o u t i n e l y s t o r e d a t - 2 0 ° . W ith in a p e r i o d
o f n o t more th a n a w eek, Super 3 was thaw ed and th e
enzyme f r a c t i o n p r e p a r e d . Super 3 was a d ju s te d to a T r i s
b u f f e r c o n c e n tr a ti o n o f 0 .1 M by th e a d d i t i o n o f 1 3 .0 ml
o f 2 M T r i s b u f f e r , pH 7 . 5 . P ro tam in e s u l f a t e s o l u t i o n
(10 mg/ml) was added u n t i l a f i n a l c o n c e n tr a ti o n o f 0 .1 7
mg o f p ro ta m in e s u l f a t e p e r m l. The p r e c i p i t a t e d p r o t a -
mine-RNA was saved f o r p r e p a r a t i o n o f r e t i c u l o c y t e RNA.
Powdered ammonium s u l f a t e was th e n added t o th e s u p e r
n a t a n t s o l u t i o n to a t t a i n ko% s a t u r a t i o n (5 8 .8 g ) , and th e
m ix tu re was c e n t r if u g e d a f t e r 1 h o u r. The p r e c i p i t a t e was
d i s c a r d e d , and ammonium s u l f a t e was added to b r in g th e
s u p e r n a ta n t to 70% s a t u r a t i o n ( 5 ^ .6 g ) . A f te r s ta n d in g
f o r 2 h o u rs w ith o c c a s io n a l s t i r r i n g , th e p r e c i p i t a t e was
c o l l e c t e d by c e n t r i f u g a t i o n f o r 30 m in u te s . The s u p e r
n a t a n t was d i s c a r d e d . The p r e c i p i t a t e was su sp en d ed i n
a p p ro x im a te ly 100 ml o f 0 .1 M T r i s b u f f e r , pH 7*5* 70%
s a t u r a t e d w ith ammonium s u l f a t e (^3*6 g o f s o l i d ammonium
s u l f a t e d i s s o l v e d i n 100 ml o f 0 .1 M T r i s b u f f e r , pH 7 * 5 ).
S u sp en sio n o f th e p r o t e i n i n t h i s s o l u t i o n p e r m itte d
rem oval o f some o f th e hem oglobin w hich p r e c i p i t a t e s a t
70# s a t u r a t i o n w ith th e enzyme f r a c t i o n . (M ost o f t h e
hem oglo bin, w hich i s p r e s e n t i n h ig h c o n c e n tr a ti o n i n
Super 3 , rem ain ed i n th e s u p e r n a ta n t a f t e r 70% ammonium
s u l f a t e p r e c i p i t a t i o n and was d i s c a r d e d ) . The suspended
p r o t e i n was a g a in c e n t r if u g e d f o r 30 m in u te s , and th e
p r e c i p i t a t e was d is s o lv e d i n 50 ml o f 0 .1 M T r i s b u f f e r ,
pH 7 . 5 , and d ia ly z e d a g a i n s t Tris-GSH-EDTA b u f f e r ( R e tic u
lo c y te f r a c t i o n AS ^ 0 - 7 0 ) . A f te r d i a l y s i s , th e enzyme
f r a c t i o n was s to r e d a t -2 0 ° i n th e p re s e n c e o f 0 .0 2 5 M GSH
and 0 .0 0 2 5 M EDTA.
R ib o n u c le ic a c i d p r e p a r a t i o n
The "pH 5 RNA" was o b ta in e d from th e pH 5 enzyme
f r a c t i o n by th e p ro c e d u re d e s c r ib e d below . A p p ro x im ately
50 ml o f pH 5 enzyme were b ro u g h t to a c o n c e n tr a tio n o f
0 .0 2 M p h o sp h a te by th e a d d i t i o n o f 1 .0 M p o ta ssiu m
p h o sp h a te b u f f e r , pH 6 . 5 . The te m p e ra tu re was k e p t a t
M - 0 . An e q u a l volume o f 90% p h en o l (8 1 , 82) was added and
th e s o l u t i o n was shaken v ig o r o u s ly i n a m e c h a n ic a l sh a k e r
f o r 1 h o u r. The e m u lsio n was c e n tr if u g e d a t 15 ,000 x g
f o r 10 m in u te s and th e aqueous (u p p e r) l a y e r was removed
w ith a s y r in g e . The p h e n o l l a y e r was m ixed th o ro u g h ly
w ith a volume o f w a te r e q u a l to th e o r i g i n a l s o l u t i o n o f
pH 5 enzym es, and a f t e r c e n t r i f u g a t i o n th e upper l a y e r was
removed and combined w ith th e f i r s t aqueous l a y e r . To th e
s o l u t i o n was added 0 .1 volume o f 20% p o ta s siu m a c e t a t e
32
and th e pH was a d ju s te d to b etw een 5 . 5 and 6 . 0 . Two
volum es o f c o ld e th a n o l were added and th e s o l u t i o n was
a llo w e d to s ta n d f o r a t l e a s t 1 h o u r and som etim es o v e r
n i g h t . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t
15*000 x g f o r 10 m in u te s , d is s o lv e d i n 5 ml o f w a te r , and
d ia ly z e d o v e r n ig h t a g a i n s t two chang es o f 2 l i t e r s e a c h o f
0 .0 2 M T r i s b u f f e r , pH 7 . 5 *
The " t r a n s f e r RNA" was p re p a re d from th e p ro ta m in e
p r e c i p i t a t e o f E lu a te 1 (se e F ig . 1 ) . The p r e c i p i t a t e o f
protamine-RNA was d is s o lv e d by h o m o g en iza tio n i n 5 to 10
ml o f 1 M p o ta ssiu m p h o sp h ate b u f f e r , pH 6 . 5 . The s o l u
t i o n was shaken w ith 90$ p h e n o l a s d e s c r ib e d b e f o r e .
A f te r c e n t r i f u g a t i o n , th e aqueous l a y e r (w hich i s th e
low er l a y e r due to th e d e n s i t y o f th e p h o sp h a te s o l u t i o n )
was rem oved. The p h e n o l l a y e r was th e n r e - e x t r a c t e d w ith
w a te r , and th e aqueous (u p p e r) l a y e r was removed and
combined w ith th e p re v io u s o n e . The combined s o l u t i o n s
were d ia ly z e d f o r a t l e a s t b h o u rs a g a i n s t b l i t e r s o f
0 .0 2 M T r i s b u f f e r , pH 7.5* a f t e r w hich p o ta s siu m a c e t a t e
was added and th e RNA re c o v e re d by e th a n o l p r e c i p i t a t i o n
and d i a l y s i s a s d e s c rib e d ab o v e. R e tic u lo c y te RNA was
p re p a re d from a protamine-RNA p r e c i p i t a t e by th e same
m ethod.
" P re in c u b a te d RNA" was p re p a re d from th e pH 5
enzyme f r a c t i o n e s s e n t i a l l y a s d e s c r ib e d by H echt e t a l .
( 8 3 ) . The pH 5 enzyme f r a c t i o n was in c u b a te d f o r 30
m in u te s a t 37° i n Medium C ( 0 .2 5 M su c ro se } 0 .0 0 5 M
magnesium c h l o r i d e ; 0 .0 2 5 M p o ta s siu m c h l o r i d e ; 0 .0 0 1 M
p o ta s s iu m p y ro p h o s p h a te ). The s o l u t i o n was th e n q u ic k ly
c h i l l e d and an e q u a l volume o f c o ld w a te r a d d e d . The
s o l u t i o n was th e n c a r e f u l l y a d ju s te d t o pH 5 .1 5 by th e
a d d i t i o n o f 1 N a c e t i c a c i d w ith c o n s ta n t s t i r r i n g . The
p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and r e
suspended i n a volume o f Medium C e q u a l t o th e o r i g i n a l
volume o f pH 5 enzymes w ith v e ry b r i e f h o m o g e n iz a tio n .
The s o l u t i o n was a g a in in c u b a te d f o r 30 m in u te s a t 3 7 ° ,
c h i l l e d w ith th e a d d i t i o n o f an e q u a l volume o f c o ld
w a te r , and th e n a d ju s te d t o pH 5 .1 5 a g a i n . A f te r c e n t r i
f u g a t i o n , th e s u p e r n a ta n t was d is c a r d e d and th e
p r e c i p i t a t e re s u sp e n d e d and hom ogenized i n 0 .1 M T r i s
b u f f e r , pH 7 . 5 . " P r e in c u b a te d RNA" was p re p a r e d from t h i
s o l u t i o n by th e same m ethod a s t h a t d e s c r ib e d f o r "pH 5
IV . EXPERIMENTAL RESULTS
S tu d ie s o f Amino A cid A c t iv a ti o n
S u b s t r a t e s o f p H 5 enzvm es-
The pH 5 enzyme p r e p a r a t i o n from g u in e a p ig l i v e r
c a t a l y z e d .t h e a c t i v a t i o n o f a l l o f th e common L-am ino
a c id s when m easured by th e PP^ exchange a s s a y (T ab le 1 ) .
( F u r th e r e v id e n c e t h a t th e lo w er PPi exchange v a lu e s
r e p o r t e d f o r a l a n i n e , a r g i n i n e , p h e n y la la n i n e , and
g lu ta m ic a c i d r e p r e s e n t " a c t i v a t i o n " o f th e s e amino a c i d s
w i l l be p ro v id e d i n a l a t e r s e c t i o n by th e f o r m a tio n o f
amino acid-RNA compounds w ith th e s e amino a c i d s i n th e
p re s e n c e o f th e pH 5 enzyme f r a c t i o n . ) A s i g n i f i c a n t
in c r e a s e o v e r th e endogenous exchange c o u ld n o t be con
s i s t e n t l y o b ta in e d w ith g lu ta m in e and h y d r o x y p r o lin e . The
PP^ exchange o b se rv e d w ith o u t th e a d d i t i o n o f amino a c i d s
i n T ab le 1 i s term ed th e endogenous e x c h a n g e . T h is r e a c
t i o n i s p ro b a b ly amino a c i d a c t i v a t i o n c a t a l y z e d by
endogenous amino a c i d s , s in c e i t i n c r e a s e d w ith tim e , was
l a b i l e to s to ra g e i n th e ab sen c e o f GSH and was i n h i b i t e d
by p - c h lo ro m e rc u rib e n z o a te a s was le u c in e a c t i v a t i o n .
Endogenous exchange was a b s e n t i n p u r i f i e d f r a c t i o n s w hich
d id n o t c o n ta in f r e e amino a c i d s . T h is d e m o n s tr a tio n o f
TABLE 1
AMINO ACID ACTIVATION BY pH 5 ENZYMES^
Amino a c id PPi_exchange
u n its/m g
L eucine .............................................. .50*+
I s o le u c in e .............................................. .^83
T ryp tophan .............................................. .216
M e t h i o n i n e ................................................... .201
Pro l i n e ........................................................ .l*+5
L ysine ........................................................ .100
T h r e o n i n e ........................................................ .09 5
S e rin e ........................................................ .075
H i s t i d in e ................................................... .068
A s p a r a g i n e ................................................... .065
A s p a r tic a c i d .............................................. .063
G ly cin e ........................................................ .063
T y r o s i n e ........................................................ .053
V alin e ........................................................ .038
A lan in e ........................................................ .016
A rg in in e . ......................................... .015
P h e n y la la n in e .............................................. .011
G lutam ic a c i d .............................................. .007
C y s t e i n e ........................................................ .007
“ F r e s h ly p re p a re d pH 5 enzymes were used i n
e ac h a s s a y . A p proxim ately 3 mg o f enzyme
were used to a s s a y th e f i r s t s i x amino
a c i d s , and 6 mg f o r th e o t h e r s . The v a lu e
i n th e absence o f added amino a c id (0 .0 3
u n i t s p e r mg) h as been s u b tr a c te d from th e
v a lu e s g iv e n above.
a c t i v a t i o n o f a l l amino a c id s i s i n ag reem en t w ith p re v io u s
r e p o r t s u sin g p ig e o n p a n c re a s (*+5) and an E . c o l l e x t r a c t
(M t). I n o th e r c a s e s i t h as n o t b een p o s s ib l e to
d e m o n s tra te th e p re s e n c e o f a l l o f th e a c t i v a t i n g enzymes
(^7s ^ 9 ) . These d i s c r e p a n c i e s a p p e a r to be due m ain ly to
th e extrem e l a b i l i t y o f many o f th e a c t i v a t i n g enzymes
w hich a r e i n a c t i v a t e d e i t h e r d u rin g p r e p a r a t i o n o r s to r a g e
o f th e e x t r a c t . The v a lu e s shown i n T able 1 , t h e r e f o r e ,
a re m inim al v a lu e s . T here was c o n s id e r a b le v a r i a t i o n i n
th e s p e c i f i c a c t i v i t i e s o f th e more l a b i l e a c t i v a t i n g
enzym es. For exam ple, th e s p e c i f i c a c t i v i t i e s f o r le u c in e
and i s o l e u c in e a c t i v a t i o n v a r ie d betw een 0 .3 5 and 0 .7 0
u n i t s p e r mg i n v a rio u s p r e p a r a t i o n s o f pH 5 enzym es. In
a d d i t i o n , some o f th e enzymes a re a c t i v e a t v e ry low con
c e n t r a t i o n s o f amino a c id such a s a re p r e s e n t i n th e pH 5
enzyme p r e p a r a t i o n and t h e i r a c t i v i t y may n o t be g r e a t l y
In c r e a s e d upon a d d i t i o n o f amino a c i d . An exam ple o f t h i s
e f f e c t i s th e v a lu e f o r v a li n e a c t i v a t i o n w hich i s o n ly
tw ic e th e endogenous exchange i n th e pH 5 enzymes (T able
1 ) , b u t v a lu e s o f 10 to 20 tim e s th e endogenous exchange
have been o b ta in e d i n E lu a te 2 where th e amino a c id
c o n te n t i s v e ry low . A lthou gh th e in c o r p o r a ti o n o f amino
a c id s i n t o th e RNA o f th e pH 5 enzyme f r a c t i o n i s
d e s c r ib e d i n d e t a i l l a t e r , i t sh o u ld be n o te d h e re t h a t
th e a c t i v i t y shown f o r any o f th e a c t i v a t i n g enzymes i s
37
p ro b a b ly s u f f i c i e n t to c a t a l y z e a maximum r a t e o f
I n c o r p o r a ti o n o f t h e c o rre s p o n d in g l a b e l e d amino a c i d
i n t o th e RNA p r e s e n t .
S t a b i l i t y o f pH 5 en zy m es.
A c tiv a tin g enzymes from g u in e a p ig l i v e r l o s t
a c t i v i t y r a p i d l y when s t o r e d a t -2 0 ° o r a t any h ig h e r
te m p e r a tu r e . The v a lu e s g iv e n i n T ab le 1 w ere o b t a i n a b l e
o n ly w ith f r e s h l y p r e p a r e d enzym es. Some o f th e enzyme
a c t i v i t i e s were d e c r e a s e d by a s much a s 75% a f t e r 2b h o u rs
o f s to r a g e i n th e f r o z e n s t a t e . I n a d d i t i o n , a l a r g e
amount o f p r o t e i n was d e n a tu r e d and p r e c i p i t a t e d from th e
pH 5 enzyme s o l u t i o n upon th a w in g . F r e e z in g and thaw ing
im m e d ia te ly d id n o t d e c r e a s e enzyme a c t i v i t y .
Many a g e n ts w ere s t u d i e d f o r p r o t e c t i o n o f th e
enzymes a g a i n s t d e n a t u r a t i o n and i n a c t i v a t i o n . S to ra g e i n
0 .0 5 M GSH c o n ta in in g 0 .0 0 5 M EDTA was th e m ost e f f e c t i v e
c o m b in a tio n f o r s t a b i l i z i n g th e a c t i v a t i n g enzymes o f th e
pH 5 f r a c t i o n . The s t a b i l i z i n g e f f e c t o f enzyme s to r a g e
i n GSH-EDTA i s shown f o r s e v e r a l enzymes i n T ab le 2 .
L y sine a c t i v a t i o n d e c r e a s e d g r e a t l y ev en w ith GSH s t o r a g e .
T h reo n in e a c t i v a t i o n was i n h i b i t e d by s to r a g e i n 0 .0 5 M
GSH, b u t was a id e d by s to r a g e i n 0 .0 1 M GSH. S to ra g e i n
GSH-EDTA d id n o t a f f e c t th e a c t i v e RNA o f th e pH 5 enzyme
w hich c o u ld s u b s e q u e n tly be rem oved by th e u s u a l p h e n o l
e x t r a c t i o n (See M eth o d s).
TABLE 2
STABILITY OF ACTIVATING ENZYMES
S to ra g e p e rio d
Amino a c id
F re s h
F ro zen 1 day
F ro zen 20 days
No GSH +gsh! /
No GSH +GSH
L eucine 1 .3 4 0 .7 9 0 1 .2 3 0 .0 3 7 0 .5 9 1
I s o le u c in e 1 .4 4
0.73*+ 1 .2 9 0 .0 27
0 .7 4 6
T y ro sin e 0 .2 0 5
— - -
0 .1 1 5
0 .2 0 0
T ry p to p h an 0 .7 0 4 - - — 0 .0 7 0
0 .3 1 5
L ysine 0 .4 2 7 0 .0 0 7 0 .0 3 3
T h reo n in e 0 .4 2 7 0 .4 8 5 0 .3 1 5
0 .2 6 4
0 .0 8 3
- S to re d i n 0 .0 5 M GSH, 0 .0 0 5 M EDTA. D ata a re PP±
exchange in u n i t s , w ith th e use o f s ta n d a rd a s s a y
c o n d itio n s w ith 3 .0 mg o f pH 5 enzym es. The a d d it i o n
o f GSH had no e f f e c t on th e a s s a y o f " f r e s h " pH 5
enzym es. The d a sh e s i n d i c a t e n o t a s s a y e d .
39
GSH-EDTA a t a lo w er c o n c e n tr a ti o n , o r 0 .0 1 M
c y s t e i n e , o r 0 .0 2 M p e n ic illa m in e were p a r t i a l l y e f f e c t i v e
i n s t a b i l i z i n g a c t i v a t i n g enzym es. O th e r s u l f h y d r y l -
c o n ta in in g compounds such a s 2 -m e rc a p to e th a n o l and t h i o -
g l y c o l l a t e were i n e f f e c t i v e o r i n h i b i t o r y . S to ra g e w ith
th e fo llo w in g a d d it i o n s d id n o t p r e s e r v e a c t i v i t y :
p o ta ssiu m b o ro h y d rid e , 0 .0 1 M magnesium c h l o r i d e , 0 .0 1 M
ATP, 10 pg o f v ita m in B ^ Pe r 0 .0 1 M p h o sp h a te b u f f e r ,
pH 7 . 5 , o r 5 mg p e r ml o f serum a lb u m in . Enzyme s t o r e d i n
O.OO^f M L - le u c in e a t -15° f o r 2 days r e t a i n e d 2 .3 tim e s a s
much a c t i v i t y f o r le u c in e a c t i v a t i o n as th e enzyme s to r e d
i n th e absence o f th e amino a c i d . T h is p r e s e r v a t i o n was
n o t a s e f f e c t i v e a s GSH-EDTA, and enzyme a c t i v i t y d e
c re a s e d much more r a p i d l y d u rin g lo n g e r p e rio d s o f
s t o r a g e . W ith more p u r i f i e d enzyme f r a c t i o n s , p a r t i c u l a r l y
F r a c t i o n AS-2, th e a d d i t i o n o f 5 mg o f serum alb u m in p e r
ml o f enzyme p lu s GSH-EDTA p ro v id e d th e m ost e f f e c t i v e
s t a b i l i z a t i o n .
P r o p e r t i e s o f p H 5 enzymes
A c t i v i t y i n th e PP^ exchange a s s a y was c o n s ta n t
f o r a t l e a s t 30 to M -0 m in u te s a t 37° f o r th e pH 5 enzym es,
a s found w ith th e p u r i f i e d t y r o s i n e - a c t i v a t i n g enzyme from
hog p a n c re a s ( 50) . PP^ exchange was p r o p o r t i o n a l to
enzyme c o n c e n tr a tio n from 0 .0 6 to 1 .5 u n i t s o f a c t i v i t y .
ho
F ig u re s 2 and 3 show th e dependence o f PP^ exchange upon
ATP and le u c in e c o n c e n tr a ti o n . Ten tim e s a s much ATP a s
amino a c id was needed f o r maximal a c t i v i t y ( F i g s . 2 , 3)*
T h is was a ls o found f o r i s o l e u c in e and th r e o n in e a c t i v a
t i o n and f o r th e p u r i f i e d t y r o s i n e a c t i v a t i n g enzyme ( 5 0 ) .
F r a c t i o n a t i o n o f th e pH 5 enzymes w ith c a lc iu m p h o sp h a te
g e l and ammonium s u l f a t e y ie ld e d enzyme f r a c t i o n s w hich
were f r e e o f endogenous amino a c id s and d id n o t c a t a l y z e
PP^ exchange i n th e ab sen ce o f added amino a c i d s . No PP^
exchange was o b se rv ed a f t e r h e a tin g th e enzyme p r e p a r a
t i o n a t 100° f o r 30 se c o n d s. No i n c o r p o r a ti o n o f P^2-
ln o r g a n ic p h o sp h ate in to ATP was c a ta ly z e d by th e pH 5
enzyme s .
The pH optimum f o r PP^ exchange showed a b ro ad
range o f maximum a c t i v i t y betw een pH 7 .5 and 9*0 w ith
l e u c i n e , i s o l e u c i n e , th r e o n i n e , t y r o s i n e , and l y s i n e .
L ysine a c t i v a t i o n was s l i g h t l y h ig h e r a t pH 7*5 b u t d id
n o t d e c re a s e g r e a t l y up to pH 8 .5 . The t y r o s i n e -
a c t i v a t i n g enzyme o f hog p a n c re a s (50) showed a pH
optimum o f 8 .5 , and th e t r y p to p h a n - a c ti v a ti n g enzyme o f
b e e f p a n c re a s (51) gave in c r e a s in g a c t i v i t y up to pH 9
i n PP^ ex ch an g e.
The a d d it i o n o f 0 .1 M p o ta ssiu m c h lo r id e
s tim u la te d ty r o s i n e a c t i v a t i o n b u t had e i t h e r no e f f e c t
o r s l i g h t l y i n h i b i t e d th e a c t i v a t i o n o f o t h e r amino a c i d s .
k l
z
v . v w
% r %
7 5
E 0.6
5
U l
O 04
□ c
U J
o .
a -
2 4 6 8 1 0
MOLES ATP/ML.
F ig u re 2 . —E f f e c t o f ATP c o n c e n tr a tio n on le u c in e
a c t i v a t i o n . S ta n d a rd a s s a y c o n d itio n s were used w ith th e
i n d ic a t e d ATP c o n c e n tr a tio n s and 3*0 mg o f s t o r e d pH 5
enzym es.
If2
^ 0.6
0
^ 0.5
V)
01
"o 0A
5 03
u J
0.2
z
0.5
>aMOLES LEUCINE/M L.
F ig u re 3 . —E f f e c t o f le u c in e c o n c e n tr a ti o n o f
le u c in e a c t i v a t i o n . S ta n d a rd a s s a y c o n d itio n s were used
w ith th e i n d i c a t e d le u c in e c o n c e n tr a tio n s and 2 .0 mg o f
s to r e d pH 5 enzym es.
T y ro sin e a c t i v a t i o n was a lm o st z e ro i n a s a l t - f r e e system
a s was fou nd w ith th e enzyme from hog p a n c re a s ( 5 0 ) .
I n h i b i t i o n o f enzyme a c t i v i t y
PP^ exchange c a ta ly z e d by th e pH 5 enzymes was n o t
i n h i b i t e d by 10 fig o f r ib o n u c le a s e p e r ml o r by 2j«moles o f
AM P p e r m l, a lth o u g h b o th o f th e s e a g e n ts c o m p le te ly i n
h i b i t e d amino a c id in c o r p o r a ti o n i n to RNA. F ig u re ^ shows
th e e f f e c t o f £ -c h lo ro m e rc u rib e n z o a te on le u c in e and
th r e o n in e a c t i v a t i o n . The r e l a t i v e i n s e n s i t i v i t y to
E -c h lo ro m e rc u rib e n z o a te o b se rv ed w ith th r e o n in e a c t i v a t i o n
( F ig . k , Curve 2) p a r a l l e l s th e enzy m e's s t a b i l i t y to
s to r a g e and i s s i m il a r to t h a t o b se rv ed w ith ty r o s i n e
a c t i v a t i o n i n th e s e s t u d i e s and w ith th e p u r i f i e d t y r o s i n e -
a c t i v a t i n g enzyme ( 5 0 ) . At a c o n c e n tr a tio n o f 2 .5 x 1 0 "1 +
M jD -c h lo ro m e rc u rib e n z o a te , le u c in e a c t i v a t i o n was in h ib ite d
90$, and th e a d d it i o n o f GSH r e s t o r e d com plete a c t i v i t y
(F ig . , Curve 1 ) . B oth enzymes were c o m p le te ly in a c -
j,
t i v a t e d by 5 x 10 M £ - c h lo r o m e r c u r ib e n z o a te , w hich a ls o
c o m p le te ly i n h i b i t e d amino a c id in c o r p o r a ti o n i n t o RNA.
I o d o a c e ta te a t 5 x 10_lf M i n h i b i t e d le u c in e and th re o n in e
a c t i v a t i o n 25 t o 35$.
P r e in c u b a tio n o f th e pH 5 enzymes w ith £ -
c h lo ro m e rc u rib e n z o a te i n th e p re s e n c e o f 1 fimole o f
l e u c i n e , o r w ith 10 pm oles o f ATP p lu s 1 0 /m o les o f
§ 0.8
z
<
□ c
H 0. 6
(JL l
£ 0 *
l u
_ 1
0 0 . 1 0.2 03 0.4 0.5
^ M O L E S PCMB /M L .
F ig u re *+.— I n h i b i t i o n o f pH 5 enzymes by jj- c h lo r o -
m e rc u rib e n z o a te (PCMB). F r e s h ly p re p a re d pH 5 enzymes
were p r e in c u b a te d a t *+° f o r 5 m in u te s w ith th e i n d i c a t e d
am ounts o f PCMB and th e n a ssa y e d i n th e s ta n d a r d way.
Curve 1 , le u c in e a c t i v a t i o n i n th e p re s e n c e o f 0 .0 5 M GSH.
The GSH was added a f t e r p r e in c u b a tio n w ith PCMB and th e
m ix tu re was p re in c u b a te d an a d d i t i o n a l 5 m in u te s b e fo re
a s s a y . Curve 2 i s th r e o n in e a c t i v a t i o n . Curve 3 i s
le u c in e a c t i v a t i o n .
magnesium c h lo r id e r e s u l t e d i n g r e a t l y d e c re a s e d
I n h i b i t i o n o f le u c in e a c t i v a t i o n (T able 3 ) - S to ra g e o f
th e pH 5 enzyme f o r s h o r t - p e r i o d s o f tim e i n 0 .0 0 k M
le u c in e a l s o a p p ea re d to p r o t e c t th e enzyme p a r t i a l l y
a g a i n s t i n a c t i v a t i o n , a s m entioned p r e v i o u s ly .
Enzyme fractionation and purification
The f r a c t i o n a t i o n shown i n F ig u re 1 h as g iv e n a
considerable purification of several of the enzymes. The
a c t i v a t i n g enzymes f o r l e u c i n e , I s o l e u c i n e , t h r e o n i n e ,
t y r o s i n e , l y s i n e , v a l i n e , and tr y p to p h a n have been s t u d i e d
in particular. The distribution and yield of the enzymes
s tu d ie d and th e p u r i f i c a t i o n s a t t a i n e d a re sum m arized i n
*
Table *+. The gel separation of threonine- and tyrosine-
activating enzymes from the other enzymes was the most
important step in obtaining these enzymes in a purified
state.
The enzyme f r a c t i o n s were f r e e d o f a c t i v e amino
a c id t r a n s f e r RNA by p r e c i p i t a t i o n o f th e RNA w ith
p ro tam in e s u l f a t e . The protamine-RNA p r e c i p i t a t e from
E lu a te 1 was th e n e x t r a c t e d w ith p h en o l to y i e l d t r a n s f e r
RNA (se e M ethods). The s u p e rn a ta n t s o l u t i o n a f t e r con
c e n t r a t i o n ( F r a c tio n AS-1) u s u a lly c o n ta in e d 30 to 60% o f
th e th r e o n in e - and t y r o s i n e - a c t i v a t i n g enzymes (T ab le h ) .
TABLE 3
EFFECT OF SUBSTRATES ON INHIBITION
BY jd -CHLOROMERCURIBENZOATE
A d d itio n s to p r e in c u b a tio n PP^ exchange
m i x t u r e ! / u n i t s
None ...................................................................... 0 .7 2
£ -C h lo ro m e rc u rib e n z o a te ......................... 0 .2 0
£ -C h lo ro m e rc u rib e n z o a te +
le u c in e .............................. O.38
E -C h lo ro m e rc u rib e n z o a te +
ATP, M g + + .................................................. 0 .3 8
£ -C h lo ro m e rc u rib e n z o a te +
l e u c i n e , ATP, Mg++ . . .. .. O.M-^
The p r e in c u b a tio n m ix tu re c o n ta in e d 2 .0 mg o f
f r e s h l y p re p a re d pH 5 enzymes and b u f f e r . The
a d d i t i o n s were th e am ounts used i n th e a s s a y .
p -C h lo ro m e rc u rib e n z o a te ( 0 .1 pm ole) was added
l a s t and th e m ix tu re p re in c u b a te d f o r * + m in u te s
a t 37°• The m ix tu re s were c o o le d to and th e
m is sin g c o n s t i t u e n t s th e n a d d ed , and a ssa y e d
a s u s u a l f o r le u c in e a c t i v a t i o n .
TABLE 4
FRACTIONATION OF pH 5 ENZYMES^/
Amino a c id
a ssa y e d
E lu a te
2
F r a c t i o n
AS-2
F r a c t i o n
AS-3
g 1 1"..... i in-
F r a c t i o n
AS-1 P u r i f i
c a t i o n
2 /
S p e c if ic
a c t i v i t y
Y ie ld S p e c if ic
a c t i v i t y
Y ie ld S p e c if ic
a c t i v i t y
Y ie ld S p e c if i c
a c t i v i t y
Y ie ld
u n its /m g
%
u n its/m g
%
u n its/m g
%
u n its /m g
%
L eucine 1 .0 2 7 4 .1
3 .4 3 *+6.2 0 .6 4
6 .9 0 0
6 .9
I s o le u c in e 0 .9 4 71 .2 2 .7 4 3 8 .6
0 .5 9
6 .6 0 0
5 .7
T ry p to p h an 0 .3 6 6 0 .6 0 .2 6 8 .2
1 .7 5 4-3.7 0 0 8 .1
L ysine 0 .1 8
65.*+ 0 .4 9 3 2 .9 0 .5 7
31.0 0 0
5 .7
T h reo n in e 0 .0 7
25.8 0 .0 1 0 .7 0 .2 3 13.0
8 .7
5 i .o 92.0
T y ro sin e
0 .0 3 2 1 .0 0 0 0 .1 8
1 8 .3 2 .9
3 0 .4 5 5 .0
V a lin e
0 .0 7 6 6 .9 0 0 0.14-
1 9 .9
0 0
3 .7
i^ E a c h f r a c t i o n was a s s a y e d i n th e s ta n d a r d way. The amount o f enzyme used was a p p r o x i
m a te ly 1 .0 u n i t o f enzyme a c t i v i t y f o r th e amino a c i d b e in g t e s t e d . The pH 5 enzyme
p r e p a r a t i o n shown i n T ab le 1 was used f o r th e f r a c t i o n a t i o n . The v a lu e s i n th e
a b sen c e o f added amino a c id s were l e s s th a n 2% o f th o s e w ith amino a c i d .
2/
— The d a ta a r e th e r a t i o o f th e s p e c i f i c a c t i v i t y o f th e b e s t f r a c t i o n to t h a t o f th e -r
pH 5 enzymes (s e e T ab le 1 ) . " < 1
*f8
E lu a te 2 was f r a c t i o n a t e d t o o b t a i n l e u c i n e - , i s o l e u c i n e - ,
l y s i n e - , and t r y p t o p h a n - a c t i v a t i n g enzym es. The d e v e lo p
ment o f t h i s f r a c t i o n a t i o n p ro c e d u re made i t p o s s ib l e to
re c o v e r b o th a c t i v a t i n g enzymes and t r a n s f e r RNA from th e
same s o u r c e , e s s e n t i a l l y f r e e o f c o n ta m in a tio n by th e
o th e r f r a c t i o n .
The AS-1 f r a c t i o n c o n ta in e d o n ly th r e o n i n e - and
t y r o s i n e - a c t i v a t i n g enzymes and was th e m ost h ig h ly
p u r i f i e d f r a c t i o n . T h r e o n in e - a c tiv a tin g enzyme had a
s p e c i f i c a c t i v i t y w hich was 90 t o 100 tim e s t h a t o f th e
pH 5 enzym es. The h ig h o v e r a l l re c o v e ry o f t h r e o n in e -
a c t i v a t i n g enzyme may r e p r e s e n t , i n p a r t , rem oval o f an
i n h i b i t o r , sin c e r e c o v e r ie s o f more th a n 100 p e r c e n t
a c t i v i t y have been o b ta in e d . The 6 - to 8 - f o ld p u r i f i c a
t i o n o f th e enzymes ir. F r a c t i o n AS-2 has been s u f f i c i e n t
f o r s t u d i e s o f a c t i v a t i n g enzymes i n r e l a t i o n t o amino
a c id i n c o r p o r a ti o n i n t o RNA d e s c r ib e d i n th e n e x t s e c t i o n .
F u r th e r p u r i f i c a t i o n o f a c t i v a t i n g enzym es, su c h a s
l y s i n e , l e u c i n e , and i s o l e u c i n e , m ight w e ll p ro ce ed from
t h i s f r a c t i o n , w h ile t r y p t o p h a n - a c t i v a t i n g enzyme was
c o n c e n tr a te d i n F r a c t i o n AS-3. F r a c t i o n AS-'t c o n ta in e d
sm a ll am ounts o f t y r o s i n e - and t h r e o n i n e - a c t i v a t i n g
enzym es. The l a b i l i t y o f th e s e enzymes p r e s e n t s a
problem w hich would make f u r t h e r p u r i f i c a t i o n d i f f i c u l t .
A ttem pts to p u r i f y v a l i n e - a c t i v a t i n g enzyme, w hich was
found i n E lu a te 2 i n good y i e l d , have b e en u n s u c c e s s fu l
due to i n a c t i v a t i o n d u rin g f u r t h e r f r a c t i o n a t i o n .
I n c o r p o r a tio n o f Amino A cids
i n t o R ib o n u c le ic A cid
The r o l e o f a c t i v a t i n g enzvmes
The i n c o r p o r a ti o n o f amino a c id s i n t o RNA h a s b een
s tu d ie d w ith a c t i v a t i n g enzyme f r a c t i o n s from w hich one o r
more o f th e a c t i v a t i n g enzymes had b een rem oved. These
f r a c t i o n s c a ta ly z e d o n ly th e in c o r p o r a ti o n i n t o RNA o f th e
amino a c i d s w hich th e y a c t i v a t e d (T able 5 )• F r a c t i o n AS-1
c a ta ly z e d th e a c t i v a t i o n o f t y r o s i n e and th r e o n in e b u t
d id n o t a c t i v a t e le u c in e o r i s o l e u c i n e , and c a ta ly z e d th e
i n c o r p o r a ti o n o f C ^ - l a b e l e d t y r o s i n e and th re o n in e in to
RNA, b u t n o t le u c in e o r i s o l e u c in e (T able 5 ). F r a c t i o n
AS-2 c a ta ly z e d th e a c t i v a t i o n o f l e u c i n e , i s o l e u c i n e , and
l y s i n e and th e i n c o r p o r a ti o n o f th e s e amino a c id s i n t o
RNA, b u t c a ta ly z e d n e i t h e r th e a c t i v a t i o n n o r th e i n
c o r p o r a tio n o f t y r o s i n e . A t r a c e o f t h r e o n i n e - a c t i v a t i n g
enzyme was p r e s e n t w hich r e s u l t e d i n a slow th re o n in e
in c o r p o r a ti o n . I t h as b een concluded from th e s e r e s u l t s
t h a t th e i n c o r p o r a ti o n o f a g iv e n amino a c id i n t o RNA
r e q u i r e s th e c o rre sp o n d in g amino a c i d - a c t i v a t i n g enzyme.
50
TABLE 5
AMINO ACID INCORPORATION INTO RNA-^
Enzyme
f r a c t i o n
C ^-A m ino a c id
I n c o r p o r a tio n
AS-1 L eucine
c .p .m .
0
I s o le u c in e 0
T h reo n in e 530
T y ro sin e 221
AS-2 L eucine 820
I s o le u c in e 29^
T h reo n in e 23
T y ro sin e 0
L ysine 710
- The a s s a y system i s d e s c r ib e d i n th e t e x t .
A p p ro x im ately 25 fig o f F r a c t i o n AS-1 o r AS-2
and 100 pg o f t r a n s f e r RNA were u se d . No
i n c o r p o r a ti o n was found w ith enzyme a lo n e
o r RNA a lo n e .
51
ILl
I n th e u s u a l in c o r p o r a ti o n a s s a y , w ith C - l e u c in e
and 30 to 60 pg o f t r a n s f e r RNA, 50 pg o f F r a c t i o n AS-2
were s u f f i c i e n t to s a t u r a t e th e RNA w ith amino a c id i n 5
m in u te s . W ith e x c e s s am ounts o f enzym e, th e a d d i t i o n o f
GSH had no e f f e c t (F ig u re 5 ) . In o r d e r to stu d y th e
r e l a t i o n s h i p b etw een PP^ exchange and amino acid-RNA
f o rm a tio n , i t was n e c e s s a r y to use l i m i t i n g am ounts o f
a c t i v a t i n g enzym e. Under th e s e c o n d it i o n s , th e enzyme was
i n a c t i v a t e d d u rin g th e i n c u b a ti o n . GSH p lu s serum alb u m in
p re v e n te d t h i s i n a c t i v a t i o n , a lth o u g h n o t c o m p le te ly
(F ig u re 5)* Serum album in a lo n e was n o t n e a r l y a s
e f f e c t i v e a s GSH a lo n e , b u t m axim al a c t i v i t y was o b ta in e d
w ith b o th p r e s e n t . W ith GSH and serum album in p r e s e n t , i t
was p o s s ib le to o b ta in a l i n e a r r a t e o f leucine-RN A
fo rm a tio n f o r th e f i r s t 6 to 9 m in u te s w ith l i m i t i n g con
c e n t r a t i o n s o f a c t i v a t i n g enzyme. The i n i t i a l r a t e o f
leucine-RN A fo rm a tio n was d i r e c t l y p r o p o r t io n a l t o enzyme
c o n c e n tr a tio n (F ig u re 6 ) . The r a t i o o f PP^ exchange to
amino acid-RNA fo rm a tio n was a p p ro x im a te ly 50 f o r l e u c i n e .
At th e lo w e s t c o n c e n tr a tio n th e enzyme was i n a c t i v a t e d
b e fo re RNA s a t u r a t i o n was re a c h e d . I t sh o u ld be n o te d
t h a t an amount o f a c t i v a t i n g enzyme, w hich s t i l l g iv e s a
s u b s t a n t i a l r a t e o f leucine-R N A f o rm a tio n (0 .0 1 to 0 .0 3
u n i t s ) , may be to o sm a ll to be d e te c te d i n th e PP^
exchange a s s a y , e s p e c i a l l y i n crud e enzyme sy ste m s.
52
? o
X
I
U
3
+GSH
NO GSH
2
1
NO GSH
0
0 5 K > 15 20 25
INCUBATION TIME IN MINUTES
30
F ig u re 5 . —E f f e c t o f GSH on leucine-R N A fo rm a tio n
a t two l e v e l s o f a c t i v a t i n g enzyme. The s ta n d a r d i n c o r
p o r a t i o n a s s a y was used w ith C ^ - l e u c i n e and *+0 pg o f
t r a n s f e r RNA. The s o l i d l i n e was o b ta in e d u sin g 100 ug o f
enzyme f r a c t i o n AS-2; p lu s 0 .0 1 M GSH ( 0 ) , m inus GSH (A ).
The d ashed l i n e was o b ta in e d u sin g 10 pg o f f r a c t i o n
AS-2} p lu s 0 .0 1 M GSH ( • ) , minus GSH (X ).
INCUBATION TIME IN MINUTES
F ig u re 6 . — E f f e c t o f a c t i v a t i n g enzyme c o n c e n tr a
t i o n on leucine-R N A f o rm a tio n . S ta n d a rd a s s a y c o n d itio n s
were used w ith C l^ - le u c in e and 80 pg o f t r a n s f e r RNA.
Curve 1 was o b ta in e d w ith 25 pg o f F r a c t i o n AS-2; Curve 2
w ith 10 pg o f F r a c t i o n AS-2; Curve 3 w ith 5 pg o f F r a c t i o n
AS-2. These am ounts o f enzyme c o rre sp o n d to 0.005* 0 .0 1 ,
and 0 .0 2 5 u n i t s o f enzyme a c t i v i t y i n a PPi exchange a s s a y
under th e same c o n d itio n s a s th e i n c o r p o r a ti o n a s s a y ,
e . g . , w ith 0 .0 2 5 pm oles o f C12- l e u c in e p r e s e n t . W ith
t h i s amount o f l e u c i n e , PPj_ exchange i s 30% o f th e m aximal
r a t e (se e F ig u re 3)*
The values shown were obtained from PP^ exchange assays at
10 to 20 times the enzyme concentrations used for incorpo
ration (Figure 6).
Assay c o n d it i o n s
The assay procedure for amino acid incorporation
into RNA was developed primarily to measure the amount of
active RNA and, therefore, other constituents were present
in excess. In early studies, the pH 5 enzyme fraction,
which contains RNA as well as activating enzymes, was used
without separation of the RNA from enzymes. In this frac
tion, the activating enzymes for leucine, isoleucine,
tyrosine, tryptophan, valine, lysine, and threonine are
present in great excess compared to the amount of RNA.
Using pH 5 enzyme or large amounts of purified enzyme plus
added RNA, maximal incorporation could be obtained with
1 x 1 0 M C^-labeled amino acid and 1 x 1 0”3 m ATP in 10
minutes (Figures 7» 3). Similar results were obtained
with tyrosine and lysine. The uniformly labeled L-leucine-
used contained a small amount of C -*-1 + - isoleucine. Thus,
1U -
depending on the level of L-leucine-C used, a variable
amount of the incorporation observed was due to C^-iso-
leucine. The data reported for C^-leucine incorporation
were obtained by assaying in the presence of excess
C^-^-isoleucine. These results have been verified by the
55
C -LEU
z
z
CM
-o
o
X
o
m /i. MOLES C * - A M I N 0 A C ID /M L .
1U
F ig u re 7 . —E f f e c t o f C # -am ino a c i d c o n c e n tr a tio n
on amino acid-RNA fo r m a tio n . S ta n d ard a s s a y c o n d itio n s
w ith th e in d ic a t e d am ounts o f C14,-am ino a c i d were u se d .
F or le u c in e i n c o r p o r a ti o n (X ), *+20 pg o f F r a c t i o n
AS-2 and 110 pg o f t r a n s f e r RNA were u se d . F or C1^ -
th r e o n in e in c o r p o r a tio n ( 0 ) , 110 pg o f F r a c t i o n AS-1 and
110 pg o f t r a n s f e r RNA were u se d .
56
6 h
s
x
%
/iMOLES ATP/ML.
F ig u re 8 . —E f f e c t o f ATP c o n c e n tr a tio n on l e u c i n e -
RNA fo r m a tio n . S ta n d a rd a s s a y c o n d itio n s w ith th e
i n d ic a t e d amounts o f ATP, 38O jig o f F r a c t i o n AS-2, and
100 o f t r a n s f e r RNA were u se d .
57
use of DL-leucine-l-C^*, a synthetically prepared isotopic
compound which does not show the C^-isoleucine contaminant.
W ith th e am ounts o f Cllf-am ino a c i d and ATP shown
to be o p tim a l f o r amino acid-RNA fo rm a tio n shown i n F ig u r e s
7 and 8 , th e a c t i v a t i n g enzymes a re n o t o p e r a tin g a t
maximal r a t e s (se e F ig u r e s 2 , 3)» b u t th e y a re p r e s e n t i n
s u f f i c i e n t e x c e ss to y i e l d m aximal r a t e s o f i n c o r p o r a t i o n .
When th e amount o f a c t i v a t i n g enzyme was re d u c e d g r e a t l y
and i n i t i a l r a t e s o f i n c o r p o r a t i o n were s t u d i e d , m aximal
r a t e s were o b ta in e d o n ly w ith 1 x 10 M amino a c i d and
0 .0 1 M ATP. These c o n c e n tr a tio n s a re s i m i l a r to th o s e
r e q u ir e d f o r maximal r a t e s o f amino a c id a c t i v a t i o n (PP.^
e x c h a n g e ). Depending on th e r a t i o o f a c t i v a t i n g enzyme to
RNA and th e l e n g t h o f tim e o f th e in c u b a ti o n , d i f f e r e n t
ATP and amino a c id c o n c e n tr a tio n s ( i n betw een th e s e l i m i t s )
would g iv e maximal i n c o r p o r a t i o n . I f th e r a t e o f i n c o r
p o r a tio n in to RNA i s slow (u n d er s u b -o p tim a l c o n d i t i o n s ) ,
th e maximal e x te n t o f l a b e l i n g may n o t be re a c h e d a t a l l
due to com peting r e a c t i o n s when cru d e e x t r a c t s a r e u se d ,
o r i n a c t i v a t i o n o f a c t i v a t i n g enzyme (s e e F ig u re 5)« Under
th e a s s a y c o n d itio n s d e s c r i b e d , m aximal i n c o r p o r a ti o n was
a c h ie v e d i n 5 to 10 m in u te s and rem ain ed a t t h a t l e v e l
(F ig u re 9)» and was p r o p o r t io n a l t o RNA c o n c e n tr a ti o n
(F ig u re 1 0 ).
58
750
S
04.
O
U -
z
4 5 0
3 0 0
on
i
U J
Z
^ 150
TIME IN MINUTES
F ig u re 9 . — Time c o u rse o f leucine-RN A f o r m a tio n .
S ta n d a rd a s s a y c o n d itio n s f o r th e i n d ic a t e d tim e p e r io d s
w ith 700 pg o f F r a c t i o n AS-2 and 80 pg o f t r a n s f e r RNA
were u se d .
59
1250
1000
750
<
z
QL
I
LU
z
500
O 250
3
£ 1
O
60 100
A 9 RNA ADDED
F ig u re 1 0 .—E f f e c t o f RNA c o n c e n tr a ti o n on
leucine-R N A fo r m a tio n . S ta n d ard a s s a y c o n d itio n s w ith th e
i n d i c a t e d amounts- o f t r a n s f e r RNA and 600 pg o f F r a c t i o n
AS-2 were u se d .
60
The optimum pH was th e same f o r th e i n c o r p o r a ti o n
o f l e u c i n e , l y s i n e , o r th r e o n in e i n t o RNA, and m aximal
r a t e s o f i n c o r p o r a tio n were o b ta in e d from pH 7 .5 t o 8 .5 .
The a d d i t i o n o f p y ro p h o sp h a ta se to th e in c u b a tio n
m ix tu re was i n d ic a t e d f o r th e rem oval o f p y ro p h o sp h a te
form ed a s a r e a c t i o n p ro d u c t (R e a c tio n 3) i n o r d e r to
p re v e n t any r e v e r s a l o f th e r e a c t i o n (8*+).
Amino a c id + ATP + RNA y a w . > ^
Amino acid-RNA + AM P + PP^
T h is a d d i t i o n doubled i n c o r p o r a ti o n in some i n s t a n c e s .
T h is e f f e c t was due to PP^ e i t h e r i n th e enzyme o r , more
u s u a l ly , i n th e RNA r a t h e r th a n PP^ p roduced d u rin g th e
r e a c t i o n . Many RNA p r e p a r a t i o n s , how ever, d id n o t show
in c r e a s e d i n c o r p o r a tio n w ith added p y ro p h o s p h a ta s e . The
b a s i s f o r th e s e d i f f e r e n c e s i s n o t y e t known.
Amino a c id t r a n s f e r r i b o n u c l e i c a c id
T able 6 shows th e d i s t r i b u t i o n o f RNA i n th e f r a c
t i o n s s t u d i e d . Only a p a r t o f th e t o t a l s o lu b le RNA, a s
i t i s u s u a lly p re p a re d , was a c t i v e f o r amino a c i d in c o rp o
r a t i o n . The g e l a b s o r p tio n and e l u t i o n w ith 2 M p h o sp h a te
b u f f e r (E lu a te 1) r e s u l t e d i n a 2 - f o l d p u r i f i c a t i o n f o r
a l l th e amino a c id s t e s t e d (T a b le s 6 and 7 ) .
TABLE 6
FRACTIONATION OF RNA
F r a c t i o n
RNA f o u n d i/
Cl1 * --. L eucine
i n c o r p o r a t i o n ^ /
mg
% %
pH 5 enzymes
3 8 .5
100 100
pH 5 RNA 3 3 .2 8 6 .0
9 6 .5
E lu a te 1
1 5 .1
3 9 .2 7 9 .8
T r a n s f e r RNA 9 .8 2 6 .M -
56.0
E lu a te 2 l*t.2 3 7 .0 1 3 .2
- The r e s u l t s a re g iv e n f o r a t y p i c a l e x p e rim e n t
s t a r t i n g w ith 130 g w et w e ig h t o f g u in e a p ig l i v e r .
The m icrosom es c o n ta in e d 170 mg o f RNA and th e
t o t a l s o lu b le RNA ( s u p e r n a ta n t a f t e r 1 ho ur a t
105»000 x g) c o n ta in e d *+8.5 mg o f RNA. The pH 5
enzymes c o n ta in e d 75% o f th e s o lu b le RNA, b u t th e
pH 5 s u p e r n a ta n t had v e ry l i t t l e a c t i v i t y f o r C ^ -
le u c in e i n c o r p o r a t i o n .
^ C -^-L eucine in c o r p o r a ti o n was m easured i n th e
s ta n d a rd a s s a y sy ste m . A c tiv a tin g enzyme (300 pg o f
F r a c t i o n AS-2) was added where n e e d e d .
62
TABLE 7
SPECIFIC ACTIVITY OF AMINO ACID-RNA COMPOUNDS
C ^-A m ino a c id
In c o r p o r a tio n :! /
P u r i f i c a t i o n
2 /
pH 5
RNA
T r a n s f e r
RNA
m|imoles/mg myunoles/mg
^L eucine 1 .6 6
3 .2 9
2 .0
I s o le u c in e 0 .8 9 1 .9 5
2 .2
L ysine
1 .7 9 2 .7 7
1 .6
T h reo n in e 0 .6 7 l.*+l 2 .1
T y ro sin e
0 .3 3
0.6*+
1 .9
l / c l b
-Amino a c id i n c o r p o r a ti o n was m easured i n th e
s ta n d a r d a s s a y sy ste m . A p p ro x im ately 300 jig o f
F r a c t i o n AS-2 f o r l e u c i n e , i s o l e u c i n e , and l y s i n e
i n c o r p o r a t i o n , 100 pg o f F r a c t i o n AS-1 f o r th r e o n in e
and t y r o s i n e i n c o r p o r a t i o n , 120 jag o f pH 5 RNA and 70
^g o f t r a n s f e r RNA were used i n th e s e e x p e rim e n ts .
O ther C ^ -a m in o a c id s were in c o r p o r a te d i n t o RNA w ith
th e fo llo w in g s p e c i f i c a c t i v i t i e s i n mpmoles p e r mgs
t r a n s f e r RNA— p r o lin e * 2 .6 7 ; v a l i n e , l.*+9; tr y p to p h a n ,
1.2*+; pH 5 RNA— a r g i n i n e , 1.7*M g lu ta m ic a c i d , 1 .1 8 ;
a l a n i n e , 1.17 j p h e n y la la n in e , 0 .6 9 ; g l y c i n e , 0 .6 2 .
-^The p u r i f i c a t i o n i s th e r a t i o o f th e s p e c i f i c
a c t i v i t i e s o f t r a n s f e r RNA to pH 5 RNA.
63
E l u t i o n w ith 2 M p h o sp h a te b u f f e r (E lu a te 1)
removed l e s s th a n 50% o f th e RNA a b so rb e d on th e g e l , b u t
t h i s f r a c t i o n c o n ta in e d Q0% o f th e a c t i v e RNA w h ile th e
E lu a te 2 f r a c t i o n c o n ta in e d l i t t l e a c t i v e RNA (T able 6 ) .
T h is f r a c t i o n a t i o n p ro b a b ly i s due to th e i n s o l u b i l i t y o f
i n a c t i v e RNA i n 2 M p h o sp h a te s i n c e , when pH 5 RNA was
f r a c t i o n a t e d w ith ammonium s u l f a t e , a f r a c t i o n o f low
a c t i v i t y was p r e c i p i t a t e d a t 65% s a t u r a t i o n a t n e u t r a l
pH ( 8 5 ) . RNA p re p a re d from g u in e a p ig l i v e r m icrosom es,
w hich was i n a c t i v e f o r amino a c id i n c o r p o r a ti o n ( 6 ^ ) , a ls o
p r e c i p i t a t e d under th e s e c o n d it i o n s . T hus, i t i s
p ro b a b le t h a t th e i n a c t i v e RNA was d e riv e d from microsome
breakdow n d u rin g th e p r e p a r a t i o n , o r may r e p r e s e n t th e
p o st-m ic ro so m a l RNA r e c e n t l y r e p o r te d ( 8 6 ).
S in ce th e same f r a c t i o n o f th e s o lu b le RNA was
p u r i f i e d f o r a l l th e amino a c id s t e s t e d (T able 6 ) , i t i s
l i k e l y t h a t a p a r t i c u l a r ty p e o f RNA can in c o r p o r a te amino
a c i d s . I t i s p ro p o sed t h a t t h i s a c t iv e RNA be term ed
"amino a c id t r a n s f e r RNA" o r " t r a n s f e r RNA." The name
d e n o te s th e f u n c tio n o f t h i s RNA w hich i s th e u s u a l b a s i s
f o r n o m en c la tu re o f enzymes and o th e r c e l l com ponents.
S tu d ie s w hich docum ent th e r o l e o f s o lu b le RNA a s an
i n te r m e d ia te i n p r o t e i n s y n t h e s i s w hich a c t s to t r a n s f e r
a c t i v a t e d amino a c id s i n t o th e m icrosom es o r rib o so m es
have b een rev iew ed by H oagland ( 2 6 ) . I t sh o u ld be n o te d
t h a t t h i s name does n o t im ply t h a t th e t r a n s f e r RNA
f r a c t i o n s tu d ie d h e re i s " p u r e 1 1 n o r t h a t “p u re " t r a n s f e r
RNA i s a s i n g l e m o le c u la r s p e c ie s o f RNA. The name d e n o te s
a ty p e o f RNA w ith a s p e c i f i c f u n c tio n w hich can be
s e p a r a te d from o th e r k in d s o f RNA b e cau se o f c e r t a i n
ch em ic al and p h y s i c a l f e a t u r e s w hich c h a r a c t e r i z e t h i s RNA.
F r a c t i o n a t i o n o f t r a n s f e r RNA has i n d i c a t e d t h a t f r a c t i o n s
s p e c i f i c f o r th e a tta c h m e n t o f i n d i v i d u a l amino a c id s a r e
p r e s e n t ( 8 5 ).
S p e c i f i c i t y o f amino a c i d - r i b o n u c l e i c
a c id fo rm a tio n
No s p e c ie s o r t i s s u e s p e c i f i c i t y f o r amino a c id
i n c o r p o r a ti o n was found among th e mammalian system s t e s t e d *
The i n i t i a l r a t e s o f C ^ - l e u c i n e in c o r p o r a ti o n were
s i m i l a r when F r a c t i o n AS-2 (g u in e a p ig l i v e r ) was used a s
a c t i v a t i n g enzyme w ith pH 5 RNA from g u in e a p i g , r a t , o r
dog l i v e r o r t o t a l s o lu b le RNA from r a b b i t r e t i c u l o c y t e s .
The t o t a l C ^ - l e u c i n e in c o r p o r a te d w ith th e s e RNA
p r e p a r a t i o n s was 1 .8 5 , 3 .2 0 , 0 . 7 , and 1 .9 0 mpmoles p e r mg,
r e s p e c t i v e l y . These am ounts p ro b a b ly r e p r e s e n t th e
t r a n s f e r RNA c o n te n t o f th e v a rio u s p r e p a r a t i o n s . Crude
a c t i v a t i n g enzyme f r a c t i o n s from r a b b i t r e t i c u l o c y t e s and
dog l i v e r gave s i m il a r r e s u l t s w ith g u in e a p ig l i v e r RNA.
I t h a s b e en r e p o r te d t h a t a s c i t e s tum or RNA p lu s l i v e r
a c t i v a t i n g enzymes were a c t i v e f o r amino a c id
65
i n c o r p o r a ti o n i n t o RNA ( 6 0 ) . However, microsom e RNA from
l i v e r o r r e t i c u l o c y t e s , t u r n i p y e llo w m osaic v i r u s RNA,
and com m ercial y e a s t RNA a l l gave l e s s th a n 0 .0 5 mpmoles o f
C l^ - ie u c in e in c o r p o r a ti o n p e r mg o f RNA. A ttem p ts to
in c r e a s e I n c o r p o r a tio n i n t o m icrosom e RNA by p r e c i p i t a t i o n
o f m icrosom es a t pH 5 b e fo r e p h e n o l e x t r a c t i o n o r by
p a r t i a l a l k a l i n e d e g r a d a tio n o f th e RNA have been u nsuc
c e s s f u l .
When g u in e a p ig l i v e r a c t i v a t i n g enzymes were used
f o r in c o r p o r a ti o n o f amino a c id i n t o E . c o l i RNA, th e
i n i t i a l r a t e o f i n c o r p o r a ti o n was low (F ig u re 1 1 ) . Since
th e i n i t i a l r a t e s o f amino a c i d in c o r p o r a ti o n i n t o any o f
th e RNA p r e p a r a t i o n s from mammalian so u rc e s were s i m i l a r ,
th e low r a t e f o r th e b a c t e r i a l RNA i n d i c a t e s a d i f f e r e n c e
betw een b a c t e r i a l RNA w hich form s C ^ -le u c in e -R N A and
l i v e r RNA o f th e same t y p e . The s p e c i f i c i t y o f amino
acid-RNA fo rm a tio n m ust be c o n t r o l l e d by th e c o n f i g u r a ti o n
o f th e a c t i v a t i n g enzyme i n a d d it i o n to d i f f e r e n c e s i n th e
RNA (85) w hich have i n d i c a t e d th e p re s e n c e o f p o ly n u c le o
t i d e c h a in s s p e c i f i c f o r i n d i v i d u a l amino a c i d s .
P r o p e r t i e s o f t r a n s f e r r i b o n u c l e i c a c id
P re v io u s s t u d i e s (6 0 , 61 *, 65) have shown t h a t th e
a c c e p to r f o r " a c t i v a t e d " amino a c id s was r i b o n u c l e i c a c id
from th e s o lu b le c e l l f r a c t i o n . The e v id e n c e in c lu d e s
66
UVER R N A
E . C O L ! RNA
X
£
0 1 0 20 30 40 50 60
INCUBATION TIME IN MINUTES
F ig u re 1 1 .—R ate o f leucine-RN A f o rm a tio n w ith
t r a n s f e r RNA and E . c o l i RNA. S ta n d a rd a s s a y c o n d itio n s
f o r th e i n d ic a t e d tim e p e rio d s w ith 600 pg o f F r a c t i o n
AS-2 and 85 pg o f t r a n s f e r RNA o r 9*f pg o f E . c o l i RNA
were u se d .
p h y s i c a l and c h e m ic a l p r o p e r t i e s , and l o s s o f a c t i v i t y
upon tr e a tm e n t w ith c r y s t a l l i n e p a n c r e a t i c r lb o n u c le a s e
and o t h e r n u c le a s e s . The a b s o r p t io n sp e c tru m o f t r a n s f e r
RNA (F ig u re 12) i s c o n s i s t e n t w ith t h a t o f n u c l e i c a c i d .
The r a t i o o f a b s o r p t io n a t 260 mp to 280 mp was 2 . 0 ,
w h ile th e 260 mp t o 230 mp r a t i o was 2 .1 t o 2 . 2 . A f te r
a l k a l i n e h y d r o l y s i s , th e a b s o r p t io n a t 258 mp i n c r e a s e d
by 38 to i n s e v e r a l e x p e rim e n ts ( F ig u r e 1 2 ) . The
e x t i n c t i o n c o e f f i c i e n t a t 260 mp, £ 1 cm = 230, was
c a l c u l a t e d from th e n u c le o ti d e c o m p o s itio n c o r r e c t e d f o r
th e h y p erch ro m ia e f f e c t . T h is v a lu e was i n good a g re em e n t
w ith th e amount o f RNA found u sin g an o r c i n o l m ethod (7 7 )
and w ith th e d e te r m in a tio n o f = 7800. T h is v a lu e
was o b ta in e d a f t e r s u b t r a c t i o n o f i n o r g a n ic p h o sp h a te
l i b e r a t e d by h y d r o l y s i s f o r 7 m in u te s a t 100° i n 1 N
s u l f u r i c a c i d . The amount o f h y d ro ly z a b le p h o sp h a te was
e q u a l to a b o u t 10# o f th e t o t a l p h o sp h a te and may be
p h o sp h a te bound to th e RNA by a l in k a g e w hich r e n d e r s i t
n o n - d i a l y z a b l e .
S tu d ie s o f th e s t a b i l i t y o f t r a n s f e r RNA, a s
m easured by th e a b i l i t y to in c o r p o r a te amino a c i d s , w ere
c o n s i s t e n t w ith i t s p o l y n u c le o tid e s t r u c t u r e . No l o s s i n
a c t i v i t y was found a f t e r 2 t o 3 m onths s to r a g e a t -2 0 °
o r a f t e r d ry in g i n a vacuum . D rie d sam p les s t o r e d a t
room te m p e ra tu re were c o m p le te ly a c t i v e a f t e r 10 d a y s .
68
> -
t z
CO
<
O
£
o
0.6
0 .5
0 .4
0 .5
02
220 240 260 280 300 320
WAVELENGTH (m /A ,)
F ig u re 1 2 .—A b so rp tio n spectrum o f t r a n s f e r ENA
b e fo re and a f t e r h y d r o l y s i s . (§ ) The sp ectru m o f 20 ug
p e r ml o f t r a n s f e r ENA was o b ta in e d a t n e u t r a l pH;
(X) sp ectru m a f t e r a l k a l i n e h y d r o l y s i s . C o n d itio n s a r e
d e s c r ib e d i n th e t e x t .
T rea tm en t a t a l k a l i n e pH r e s u l t e d i n l o s s e s o f a c t i v i t y
w hich p a r a l l e l e d th e i n c r e a s e i n p e r c h l o r i c a c i d - s o l u b l e
m a t e r i a l . When t r a n s f e r RNA was h e a te d a t te m p e r a tu re s
up to 70° a t pH 6 .8 i n 0 .0 2 M T r i s b u f f e r , no l o s s i n
a c t i v i t y was fo u n d . H e a tin g a t 80° f o r 80 m in u te s o r a t
100° f o r M -5 m in u te s r e s u l t e d i n l o s s o f 50# o f th e
a c t i v i t y . T r a n s f e r RNA, w hich was 70# i n a c t i v a t e d a f t e r
h e a tin g a t 1 0 0 °, showed no in c r e a s e i n a b s o r p t io n a t 260
mp and was s t i l l a c i d - p r e c i p i t a b l e . T h is f in d in g su g g e ste d
t h a t l i t t l e o r no d e g ra d a tio n o f th e m o le cu le had
o c c u rre d . The h e a t i n a c t i v a t i o n may have been due to
a g g r e g a tio n s in c e a g r e a t e r p r o p o r tio n o f th e h e a te d RNA
was p r e c i p i t a t e d by ammonium s u l f a t e th a n th e u n h eated
c o n t r o l . No d i f f e r e n c e i n th e r a t e o f i n a c t i v a t i o n a t
100° was found f o r l e u c i n e , t h r e o n i n e , and l y s i n e i n c o r
p o r a t i o n .
The s t a b i l i t y o f t r a n s f e r RNA to h e a tin g i s
g r e a t e r th a n to b ac co m osaic v i r u s RNA ( 8 8 ) , b a se d on b i o
l o g i c a l a c t i v i t y and p h y s i c a l p r o p e r t i e s . T h is d i f f e r e n c e
i s p ro b a b ly p a r t i a l l y due to th e much l a r g e r s iz e o f
to b ac co m osaic v i r u s RNA. The h y p erch ro m ic change o f
t r a n s f e r RNA w ith i n c r e a s e d te m p e ra tu re i n d i c a t e s th e
p re s e n c e o f some seco n d ary s t r u c t u r e w hich may be o f g r e a t
im p o rtan ce f o r b i o l o g i c a l a c t i v i t y . F u r th e r s t u d i e s o f
h e a t - i n a c t i v a t e d t r a n s f e r RNA sh o u ld p ro v id e in f o r m a tio n
on this problem.
i
S tu d ie s o f th e amino a c i d -
r i b o n u c l e i c a c i d lin k a g e
In r e p o r t s from two l a b o r a t o r i e s , th e amino a c i d -
RNA lin k a g e h a s been fo rm u la te d a s an e s t e r betw een th e
c a rb o x y l o f th e amino a c i d and a 2 ' - o r 3*-h y d ro x y l o f th e
r ib o s e o f a t e r m in a l a d e n o sin e ( 8 3 , 89 ) . R e s u lts o b ta in e d
by C ordes i n o ur l a b o r a t o r y (73) i n d i c a t e d t h a t t r a n s f e r
RNA te r m in a te s i n AMP a t one end and may n o t have a
unique te rm in u s a t th e o t h e r e n d . The d a ta showed a r a t i o
o f a p p ro x im a te ly 120 n u c le o ti d e s p e r a d e n o s in e , which
would r e p r e s e n t th e a v e ra g e m o le c u la r w e ig h t i f a l l th e
c h a in s p r e s e n t te r m in a te d i n a d e n o s in e . T h is would be th e
maximum m o le c u la r w e ig h t s in c e m ost c o n ta m in a n ts would be
o f much h ig h e r m o le c u la r w e ig h t, and i f th e y te r m in a te d i n
n u c le o s id e - 3 '- p h o s p h a te would n o t be d e t e c t e d . A lso ,
sin c e a d e n o sin e was such a m inor c o n s t i t u e n t o f th e
m ix tu re ( 0 .77% ), some l o s s e s u n d o u b ted ly o c c u rre d ( 7 3 ) .
A r a t i o o f th e number o f n u c le o ti d e s i n t r a n s f e r RNA p e r
amino a c id in c o r p o r a te d may be c a l c u l a t e d from th e a v e ra g e
in c o r p o r a ti o n o f th e e i g h t C ^ - l a b e l e d amino a c i d s t e s t e d
(se e T ab le 7 ) . The v a lu e i s b a sed on th e a ssu m p tio n t h a t
a l l e ig h te e n amino a c id s would be in c o r p o r a te d to th e same
e x t e n t . T h is r a t i o i s 82 and a ls o r e p r e s e n t s a p o s s ib l e
maximum s i z e f o r th e RNA c h a in .
71
The f i n d i n g s t h a t t r a n s f e r RNA te r m in a te d I n AMP,
and t h a t amino a c i d I n c o r p o r a t i o n was a p p ro x im a te ly e q u a l
on a m o lar b a s i s t o th e a d e n o s in e form ed by a l k a l i n e
h y d r o l y s i s , p ro v id e a d d i t i o n a l e v id e n c e t h a t a l l th e amino
a c i d s a r e l in k e d to a t e r m in a l AMP w hich was p r e s e n t on
t r a n s f e r RNA a s i s o l a t e d . S tu d ie s o f th e i n c o r p o r a t i o n
o f C ^-A T P i n t o RNA (90) have c o n firm e d th e s e f i n d i n g s .
In th e c o u rs e o f th e work p r e s e n te d h e r e , enzym es w hich
c a ta ly z e d i n c o r p o r a t i o n o f th e C^-AMP o f ATP i n t o RNA
w ere foun d i n F r a c t i o n A S-1, b u t n o t I n o t h e r f r a c t i o n s ,
su c h as F r a c t i o n AS-2. P re in c u b a te d RNA, w hich had l o s t
te r m in a l n u c le o ti d e s (S 3 ), d id n o t in c o r p o r a te amino a c id s
u n le s s te r m in a l n u c l e o t i d e s were r e p la c e d u sin g F r a c t i o n
AS-1 (T ab le 8 ) . On th e o t h e r h a n d , t r a n s f e r RNA showed
m aximal amino a c i d i n c o r p o r a t i o n w ith F r a c t i o n AS-2 a lo n e
and amino a c id i n c o r p o r a t i o n was n o t i n c r e a s e d by th e
a d d i t i o n o f F r a c t i o n A S-1.
P re in c u b a te d RNA, w hich h as l o s t i t s te r m in a l AM P
and p o s s ib l y CMP ( 83 ) , was t e s t e d w ith enzyme f r a c t i o n s
AS-1 and AS-2. F r a c t i o n AS-2, w hich c o n ta in s l e u c i n e -
a c t i v a t i n g enzym e, d id n o t c a t a l y z e ClLf-ATP i n c o r p o r a t i o n
i n t o p re in c u b a te d RNA, and t h i s RNA was i n a c t i v e f o r
c l ^ - i e u c i n e i n c o r p o r a t i o n (T ab le 8 ) . Cllf- i s o le u c in e was
a l s o n o t I n c o r p o r a te d under th e s e c o n d i t i o n s . F r a c t i o n
AS-1, w hich c o n ta in s t h r e o n i n e - a c t i v a t i n g enzym e,
72
TABLE 8
Clif-ATP AND Cllf-AMINO ACID INCORPORATION
INTO PREINCUBATED RNAl/
A d d itio n s
Enzyme
f r a c t i o n
C ^-A T P
in c o rp o
C ^-A m in o a c i d
i n c o r p o r a t i o n
r a t i o n
mpmoles
C1^ -
th r e o n in e
mpmoles
C ^ -
le u c in e
mpmoles
Com plete
system AS-2 0
w a .
0
Com plete
system
+ CTP AS-2 0 .0 0 1 *
-
0 . 00* f
Com plete
system AS-1 0.76 0 .0 0 5
0
Com plete
system
+ CTP AS-1 1 .9 0 0 .0 7 5
0.00 1
Com plete
system
AS-2 +
AS-1 0.8 0 0.008 0.090
Com plete
system
+ CTP
AS-2 +
AS-1
1 .9 8 0 .0 7 5 0 .1 9 ^
” C-^-ATP and C ^ -a m in o a c i d i n c o r p o r a t i o n were m easured
i n th e s ta n d a rd a s s a y s y s te m s . CTP, 0 .1 pm ole, was
added w here i n d i c a t e d . A p p ro x im ately 100 jag o f
F r a c t i o n A S-1, 600 jig o f F r a c t i o n A S-2, a n d 100 jig o f
p r e in c u b a te d pH 5 RNA w ere u s e d . I n th e C-^-ATP
i n c o r p o r a t i o n system u sin g 100 jug o f F r a c t i o n A S-1,
0 .2 3 mjimole o f cl^-A T P were in c o r p o r a te d i n th e
a b se n c e o f RNA o r when F r a c t i o n AS-1 was p r e c i p i t a t e d
w ith t r i c h l o r o a c e t i c a c i d a t z e ro tim e . T h is v a lu e was
c o n s id e r e d a n o n -e n z y m a tic a b s o r p t i o n o f r a d i o a c t i v i t y
and h a s b een s u b t r a c t e d from v a lu e s i n th e t a b l e s .
( L a te r enzyme p r e p a r a t i o n s gave no Cl^-ATP i n c o r p o r a
t i o n i n th e a b sen c e o f added RNA.) D ashes i n d i c a t e
n o t t e s t e d .
73
c a t a l y z e d C-^-ATP i n c o r p o r a t i o n i n t o p r o in c u b a te d RNA.
T h is i n c o r p o r a t i o n was d o u b le d i n th e p re s e n c e o f un
l a b e l e d CTP, and Cllf- t h r e o n i n e was i n c o r p o r a te d to a
m axim al e x t e n t i f CTP w ere p r e s e n t . T hese r e s u l t s demon
s t r a t e t h a t F r a c t i o n AS-2 l a c k s th e enzymes f o r C ^-A T P
i n c o r p o r a t i o n and t h a t p r e in c u b a tio n h a s c o m p le te ly i n a c
t i v a t e d th e RNA a s shown by th e a b sen c e o f amino a c id
i n c o r p o r a t i o n . R e p a ir o f th e same RNA was shown by th e
i n c o r p o r a t i o n o f C ^ " - th r e o n in e i n th e p re s e n c e o f CTP,
u sin g F r a c t i o n A S-1, w hich c a ta ly z e d C ^-A T P i n c o r p o r a t i o n .
When F r a c t i o n s AS-2 and AS-1 were com bined, C ^ - l e u c i n e
and C l ^ - i s o l e u c i n e i n c o r p o r a t i o n was a l s o r e s t o r e d , th u s
c o n firm in g th e c o n c lu s io n t h a t re p la c e m e n t o f th e AM P
m o ie ty was r e q u i r e d f o r amino a c id i n c o r p o r a t i o n . The
114 -
a d d i t i o n o f CTP d o u b led th e i n c o r p o r a t i o n o f C -ATP and
Cllf- l e u c i n e (T ab le 8 ) . A d d itio n o f PPj^ had no e f f e c t on
cl^-A TP i n c o r p o r a t i o n w ith p r e in c u b a te d RNA. H igher
l e v e l s o f PP^ have b een r e p o r t e d to i n h i b i t t h i s r e a c t i o n
( 9 0 ) . These r e s u l t s a re c o n s i s t e n t w ith th e f o r m u la tio n
o f th e te rm in u s f o r amino a c i d a tta c h m e n t a s : RNA-pCpCpA
( 83 ) , and i n d i c a t e t h a t p r e i n c u b a t i o n h as rem oved AMP from
a l l th e c h a in s and CM P from h a l f th e c h a i n s . In o th e r
e x p e r im e n ts , CTP d o u b le d th e i n c o r p o r a t i o n o f C ^ -
i s o l e u c i n e , a l s o . The 1 0 - f o ld s t i m u l a t i o n o f C ^ -
th r e o n in e i n c o r p o r a t i o n by CTP (T ab le 8 ) , how ever,
7^
s u g g e s ts t h a t a lm o s t a l l th e CMP was removed from th e RNA
s p e c i f i c f o r th r e o n in e i n c o r p o r a t i o n ( 8 5 ) . A d d itio n a l
p r e p a r a t i o n s o f p r e in c u b a te d RNA have b een made by
r e p e a te d in c u b a ti o n o f th e pH 5 enzymes w ith PP^ (se e
M ethods) i n w hich th e i n c o r p o r a t i o n o f C ^-A T P and th e
l U -
i n c o r p o r a t i o n o f a l l o f th e Cx -am ino a c i d s t e s t e d was
a lm o s t c o m p le te ly d e p e n d e n t on th e a d d i t i o n o f CTP.
When t r a n s f e r RNA p r e p a r a t i o n s were t e s t e d i n th e
same way, c o m p le te ly d i f f e r e n t r e s u l t s were o b ta in e d .
I n c o r p o r a ti o n o f C ^-A T P was c a ta ly z e d by F r a c t i o n AS-1
and n o t by F r a c t i o n A S-2, b u t was a lm o s t c o m p le te ly
d e p e n d e n t on PP^ a d d i t i o n . CTP a d d i t i o n a t 1 x 10"^ M
had no e f f e c t i n c o n t r a s t to r e s u l t s o b ta in e d w ith p r e -
ln c u b a te d RNA. The a d d i t i o n o f 0 .0 0 1 M CTP i n h i b i t e d
c l 1 * -a tP i n c o r p o r a ti o n by 50 to 75%* The PP^ re q u ire m e n t
f o r i n c o r p o r a t i o n o f C ^ -A T P i n t o t r a n s f e r RNA i s i n
a g reem en t w ith th e f u n c t i o n o f PPj^ i n th e rem oval o f
te r m in a l n u c l e o t i d e s from RNA ( 9 0 ) .
Maximal i n c o r p o r a t i o n o f C - ^ - le u c in e and C ^ -
i s o l e u c i n e i n t o t r a n s f e r RNA was o b ta in e d w ith F r a c t i o n
AS-2 a lo n e . The a d d i t i o n o f F r a c t i o n AS-1 ( c o n ta in in g
Cl^-ATP i n c o r p o r a ti n g enzyme) and CTP had no e f f e c t .
These r e s u l t s i n d i c a t e t h a t t r a n s f e r RNA, a s i s o l a t e d ,
c o n ta in e d a l l o f i t s te r m in a l AMP. Some C ^-A T P in c o r p o
r a t i o n i n t o t r a n s f e r RNA was o b se rv e d i n th e ab sen c e o f
75
added PP^ and v a r i e d I n amount w ith d i f f e r e n t p r e p a r a t i o n s
o f t r a n s f e r RNA, T h is i n c o r p o r a t i o n may be due to
v a r i a b l e am ounts o f endogenous PP^^ i n th e RNA p r e p a r a t i o n ,
o r a v e ry sm a ll p e rc e n ta g e o f th e t r a n s f e r RNA may l a c k
t e r m in a l AMP. The C ^-A T P i n c o r p o r a t i o n o b se rv e d w ith
t r a n s f e r RNA a p p e a rs to be due to an exchange r e a c t i o n .
The t e r m in a l AMP i s removed a s R e a c tio n * + p r o c e e d s , and
r e v e r s a l r e s u l t s i n i n c o r p o r a t i o n o f C ^-A T P i n t o RNA.
RNA-pCpCpA + PPi RNA-pCpC + ATP (*+)
The p r e p a r a t i o n o f a c t i v a t i n g enzymes f r e e from
th e enzymes w hich c a t a l y z e th e a d d i t i o n o f end g ro u p s to
RNA i n d i c a t e s t h a t th e l a t t e r r e a c t i o n s a re n o t d i r e c t l y
r e q u i r e d f o r th e i n c o r p o r a t i o n o f amino a c i d s i n t o RNA
when th e RNA i s i s o l a t e d und er th e c o n d itio n s d e s c r ib e d
h e r e .
F u r t h e r in f o r m a tio n on th e n a tu r e o f th e amino
acid-RNA lin k a g e h a s b een o b ta in e d from a s tu d y o f th e
r e v e r s i b i l i t y o f R e a c tio n 3 (F o rm a tio n o f Amino acid-R N A ).
The a d d i t i o n o f AMP a f t e r m axim al i n c o r p o r a t i o n was
r e a c h e d , r e s u l t e d i n a breakdow n o f threonine-R N A (F ig u re
1 3 ) . S im ila r r e s u l t s have b een r e p o r t e d by o t h e r w o rk e rs
(6 0 , 6 5 , 9 1 ) . However, su ch r e s u l t s do n o t e x c lu d e
h y d r o l y s i s and o t h e r ty p e s o f l o s s e s o f amino acid-RNA.
The f a c t t h a t AMP added i n i t i a l l y gave th e same f i n a l
76
§
S
£
<
z
400
•A
300
V AMP AT 5 M/NUTES
OC 200
u i
Z
Z
s
c *
£
o-
100
20
TIME IN MINUTES
F ig u re 1 3 .—E f f e c t o f AM P on threonine-R N A
f o rm a tio n . The s ta n d a rd a s s a y c o n d itio n s f o r th e
i n d ic a t e d tim e p e rio d s were u se d . (6) S ta n d a rd a s s a y ,
w ith o u t AMP; (0) 2 pm oles o f AMP added a f t e r 5 m in u te s
in c u b a tio n ; ( t ) 2 pm oles o f AMP added i n i t i a l l y .
amount o f threonine-R N A i s e v id e n c e t h a t th e r e a c t i o n i s
r e v e r s i b l e and t h a t th e amount o f threonine-R N A found
a f t e r AM P a d d i t i o n r e p r e s e n t s th e e q u ilib r iu m p o s i t i o n
under th e s e c o n d i t i o n s . O ther e x p e rim e n ts w hich s u p p o rte d
t h i s c o n c lu s io n were th e f a i l u r e o f AMP to produce a l o s s
o f threonine-R N A i n th e p re s e n c e o f p y ro p h o s p h a ta s e ,
com plete i n h i b i t i o n o f th e AM P e f f e c t when th r e o n in e -
a c t i v a t i n g enzyme was i n h i b i t e d w ith 0 .0 0 1 M £ - c h lo r o -
m e rc u rib e n z o a te , and p r o p o r t i o n a l i t y o f threonine-RN A
c le a v e d to th e amount o f AMP ad d ed . These r e s u l t s have
been confirm ed by th e d e m o n s tra tio n o f c le a v a g e o f
i s o l a t e d threonine-R N A i n th e absence o f ATP and th e i n
c o r p o r a tio n o f C^-AMP in to ATP ( 8^-). The amino acid-RNA
lin k a g e t h e r e f o r e f a l l s i n t o th e g e n e r a l c l a s s i f i c a t i o n o f
"h ig h e n erg y " bonds ( 8*+).
E f f e c t s o f i n h i b i t o r s on amino a c i d -
r i b o n u c l e i c a c id f o rm a tio n
The i n h i b i t i o n o f leucine-RNA fo rm a tio n by
£ -c h lo ro m e rc u rib e n z o a te and io d o a c e ta te p a r a l l e l e d th e
e f f e c t s o f th e s e i n h i b i t o r s on le u c in e a c t i v a t i o n
m entioned e a r l i e r . W ith F r a c t i o n AS-2 a s a c t i v a t i n g
enzyme, C ^ - l e u c i n e in c o r p o r a ti o n was i n h i b i t e d 95 to 100#
by 5 x 10"5 m £ -c h lo ro m e rc u rib e n z o a te and o n ly 17# by
0 .0 0 1 M io d o a c e ta m id e . GSH was o m itte d from a s s a y s w hich
c o n ta in e d t h i o l i n h i b i t o r s . H igher l e v e l s o f
jj-c h lo ro m e rc u rib e n z o a te were needed f o r i n h i b i t i o n o f
i n c o r p o r a ti o n u sin g pH 5 enzyme, p resum ably b ecau se o f
endogenous g ro u p s w hich c o u ld b in d th e i n h i b i t o r .
l y s i n e i n c o r p o r a ti o n u sin g F r a c t i o n AS-2 was i n h i b i t e d
b-7% w ith 2 .5 x 10”^ M £ - c h lo ro m e rc u r ib e n z o a te . C^*-
th r e o n in e in c o r p o r a ti o n was i n h i b i t e d 75% by 5 x 10"^ M
£ -c h lo ro m e rc u rib e n z o a te and showed l i t t l e o r no i n h i b i t i o n
w ith 0 .0 0 1 M io d o a c e ta m id e u sin g F r a c t i o n AS-1 a s a so u rc e
o f t h r e o n i n e - a c t i v a t i n g enzyme. These r e s u l t s a re con
s i s t e n t w ith th e g r e a t e r r e s i s t a n c e o f th r e o n in e -
a c t i v a t i n g enzyme to th e s e i n h i b i t o r s (se e F ig u re *+).
The i n h i b i t o r y e f f e c t o f PP^ on C ^ - l e u c i n e
i n c o r p o r a ti o n usin g F r a c t i o n AS-2 was p r o p o r t io n a l to th e
c o n c e n tr a tio n and 50% i n h i b i t i o n was o b ta in e d a t 1 .5 x
1 0 M P P ^
1 lx
When s e v e r a l C -am ino a c id s were t e s t e d t o g e t h e r ,
th e i n c o r p o r a ti o n was a d d i t i v e . T his f in d in g su g g e ste d
t h a t RNA c h a in s s p e c i f i c f o r i n d i v i d u a l amino a c id s were
p r e s e n t i n t r a n s f e r RNA and le d to th e f r a c t i o n a t i o n
s t u d i e s by Sm ith, C o rd e s, and Schweet w hich have p ro v id e d
e v id e n ce f o r t h i s v ie w p o in t (6M-, 8 5 ). The a d d i t i v i t y o f
i n c o r p o r a ti o n w ith s e v e r a l C ^ -a m in o a c id s has b een con
firm e d w ith le u c in e p lu s i s o l e u c i n e , th r e o n i n e , l y s i n e ,
o r v a l i n e ; and i s o l e u c i n e p lu s v a l i n e , u sin g s ta n d a rd
a s s a y c o n d it i o n s . As e x p e c te d from th e s e r e s u l t s , th e
79
a d d i t i o n o f n o n - r a d i o a c t iv e i s o l e u c i n e , l y s i n e , th r e o n i n e ,
o r v a li n e d id n o t i n h i b i t C11+- l e u c in e i n c o r p o r a t i o n .
Amino Acid-Ribonucleic Acid Formation
as an Intermediate Step Tn
Protein Synthesis
The synthesis of hemoglobin in a
cell-free system
The m icrosom al p a r t i c l e s have been d e s ig n a te d as
th e m ajor s i t e s o f p r o t e i n s y n th e s is w ith in th e c e l l s o f a
v a r i e t y o f t i s s u e s . S tu d ie s w ith i n t a c t a n im a ls and
v a r io u s ty p e s o f whole c e l l sy s te m s, rev iew ed by Askonas
e t a l . (92) a s w e ll a s th e work o f P alad e (93) w ith th e
e l e c t r o n m ic ro sc o p e , p ro v id e d th e o r i g i n a l e v id e n ce f o r
t h i s c o n c lu s io n . The term s "m icrosom al p a r t i c l e s " o r
"m icrosom es" a r e now g e n e r a l l y used to d e s c r ib e th e
m icrosom es o f t i s s u e s such a s l i v e r , and c o n s i s t o f r i b o -
n u c le o p r o te in p a r t i c l e s a tta c h e d to th e en d o p lasm ic
r e tic u lu m . The r ib o n u c l e o p r o t e i n p a r t i c l e s o f t i s s u e s
such a s r e t i c u l o c y t e s , i n w hich no en d o p lasm ic r e tic u lu m
i s e v id e n t, have been named "rib o so m es" f o r d i s t i n c t i o n .
The in te r m e d ia te r e a c t i o n s in v o lv e d i n p r o t e i n
s y n th e s is have o n ly r e c e n t l y become a p p ro a c h a b le a s
ch em ical p ro b le m s. A s i g n i f i c a n t e v e n t w hich in f lu e n c e d
t h i s change i n a p p ro a c h was th e f in d in g o f Zam ecnik and
K e lle r (3 ^ ) t h a t a c t i v e system s f o r th e in c o r p o r a ti o n o f
80
C ^ -a m in o a c i d s i n t o p r o t e i n c o u ld be c e n t r i f u g a l l y
f r a c t i o n a t e d i n t o two e s s e n t i a l c e l l u l a r com ponents—
m icrosom es and s o lu b le enzym es. A lth ough th e c e l l - f r e e
p r e p a r a t i o n s d ev elo p ed i n r e c e n t y e a rs a r e a l l in a d e q u a te
f o r com plete s tu d y , i t h as begun to a p p e a r t h a t th e p a t
t e r n o f r e a c t i o n sequence i s s i m i l a r i n b a c t e r i a l (*+0 , 65 ) ,
p ro to z o a n (91 ) , h ig h e r p l a n t ( 95 )* a v ia n (67 ) , and
mammalian t i s s u e s (M-3, 6 0 , 6*+).
In o r d e r to i n v e s t i g a t e th e r e l a t i o n s h i p betw een
amino acid-RNA fo rm a tio n and p r o t e i n s y n t h e s i s , i t was
n e c e s s a r y to d e v elo p te c h n iq u e s f o r th e p r e p a r a t i o n o f a
c e l l - f r e e system w hich would in c o r p o r a te amino a c id s i n to
p r o t e i n . The s i g n i f ic a n c e o f a l l s t u d i e s on th e in c o r p o
r a t i o n o f C ^ -a m in o a c id s i n t o p r o t e i n i n c e l l - f r e e
p r e p a r a t i o n s , how ever, depends i n th e l a s t a n a l y s i s on
w h eth er th e i n c o r p o r a ti o n r e p r e s e n t s a f r a c t i o n o f th e
t r u e i n v iv o p r o c e s s o f p r o t e i n s y n t h e s i s . Hoagland (2k)
has c o n c lu d e d , i n re v ie w in g th e r e s u l t s o f many w orkers v i n
t h i s f i e l d , t h a t t h i s c r i t e r i o n would be m ost s a t i s
f a c t o r i l y met i n a system w hich c o u ld be f r a c t i o n a t e d i n t o
i t s i n d i v i d u a l e n z y m a tic , p a r t i c u l a t e , and n u c le o tid e
com ponents and s t i l l be c a p a b le o f b r in g in g ab o u t
s y n th e s is o f i s o l a t a b l e p r o t e i n , i d e n t i f i a b l e a s s p e c i f i c
to th e t i s s u e o f o r i g i n . Kruh and B orsook ( 96 ) have
d e m o n s tra te d t h a t r a b b i t r e t i c u l o c y t e s s y n th e s iz e
81
hem oglobin i n v i t r o and t h a t a p p ro x im a te ly 85# o f th e
s o lu b le p r o t e i n made by su ch c e l l s i s o f t h i s one s p e c i e s .
S tu d ie s w ith whole r e t i c u l o c y t e c e l l s by R a b in o v itz and
O lson (97) have shown t h a t rib o so m es p a r t i c i p a t e i n th e
s y n t h e s i s o f h em o glo bin . These a u th o r s have a ls o demon
s t r a t e d t h a t th e I n c o r p o r a ti o n o f i r o n i n t o hem oglobin
ta k e s p la c e i n a c e l l - f r e e system c o n ta in in g r e t i c u l o c y t e
rib o so m es (9 8 ). I t was c l e a r , th e n , t h a t r e t i c u l o c y t e s
o f f e r e d a f a v o r a b le system f o r stu d y o f th e s y n th e s is o f a
s p e c i f i c p r o t e i n . The p r e p a r a t i o n o f a c e l l - f r e e system
from r e t i c u l o c y t e s w hich would produce hem oglobin was n o t
th e p rim a ry o b j e c ti v e o f th e r e s e a r c h problem s t a t e d i n
t h i s d i s s e r t a t i o n , b u t e a r l y r e s u l t s (99) proved so
p ro m isin g t h a t a g r e a t d e a l o f r e s e a r c h e f f o r t has been
expended i n th e developm ent o f th e sy stem . T h e r e fo r e ,
o n ly th e d e t a i l s c o n c e rn in g th e system w hich a re e s s e n t i a l
i n u n d e rs ta n d in g th e p a r t i c i p a t i o n o f in te r m e d ia te r e a c
t i o n s a re p r e s e n te d h e r e .
P r o p e r t ie s o f th e amino a c id
in c o r p o r a ti o n system
The r e s u l t s shown i n T able 9 show th e in c o r p o r a
t i o n o f Cllf- l e u c in e i n t o p r o t e i n , u sin g rib o so m es p re p a re d
from r a b b i t r e t i c u l o c y t e s . The enzyme used was R e t i c u l o
c y te F r a c t i o n AS ^ 0 -7 0 w hich was r i c h i n amino a c id
a c t i v a t i n g enzym es, and c a ta ly z e d r a p i d amino acid-RNA
82
TABLE 9
INCORPORATION OF Cll+-LEUCINE INTO PROTEIN
R e a c tio n m ix tu re c .p .m ./m g
rib o so m es
Com plete sy ste m ^ / ............................................ 1722
Complete sy stem , m inus ATP,
m inus A T P -gen erating system .................... 9
Complete sy stem , m inus ribosom es . . . 0
Complete sy stem , m inus enzyme f r a c t i o n . 165
Complete sy stem , m inus amino
a c id m ix tu re . 71 +3
Complete sy stem , m inus RNA ( s o lu b le ) . . 739
Complete sy stem , m inus GSH .......................... 792
Complete sy stem , m inus magnesium
c h lo r id e ................................................................ 82
“ The com plete system c o n ta in e d th e s ta n d a r d a s s a y
c o n s t i t u e n t s , w ith 5 mg o f rib o so m e s , 2 mg o f
R e tic u lo c y te F r a c t i o n AS *f0-70, and 5 5 fig o f
r e t i c u l o c y t e RNA. The in c u b a tio n tim e was ^-0
m in u te s .
f o r m a tio n w ith added t r a n s f e r RNA. V ery l i t t l e a c t i v e
t r a n s f e r RNA rem ain ed i n t h i s enzyme f r a c t i o n when
p r e p a r e d a s d e s c r ib e d i n th e s e c t i o n on m eth o d s. The
p r o c e s s o f amino a c i d i n c o r p o r a t i o n shown i n T ab le 9 i s
v e ry e n e rg y -d e p e n d e n t and r e q u i r e s m agnesium i o n , a s
o r i g i n a l l y o b se rv e d by Zam ecnik and K e l le r (3*+), u s in g r a t
l i v e r m icro so m es. The s u b s t i t u t i o n o f 10 pm oles o f ATP
f o r th e A T P -g e n era tin g system ( c r e a t i n e p h o sp h a te and ATP-
c r e a t i n e tr a n s p h o s p h o r y la s e ) i n h i b i t e d i n c o r p o r a t i o n
a lm o st c o m p le te ly . No i n c o r p o r a t i o n o c c u rr e d i n th e
ab sen ce o f rib o s o m e s , w ith b o i l e d rib o s o m e s , o r w ith c e l l
d e b r i s s u b s t i t u t e d f o r rib o s o m e s . Ribosom es s t o r e d i n
0 .2 5 M s u c ro s e a t -2 0 ° f o r 5 day s r e t a i n e d 90# o f th e
i n c o r p o r a t i o n a c t i v i t y o b se rv e d i n f r e s h l y p re p a re d r i b o
some s .
In th e a b sen ce o f GSH, 1 .0 pm oles o f p - c h l o r o -
m e rc u rib e n z o a te i n h i b i t e d i n c o r p o r a t i o n by 95#. I n th e
a b sen ce o f added RNA, 0 .1 pg o f c r y s t a l l i n e r ib o n u c le a s e
re d u c e d I n c o r p o r a ti o n by 80# .
The a d d i t i o n o f th e co m plete amino a c id m ix tu re
d e v elo p ed by B orsook and co w ork ers (7 ^ ) in c r e a s e d th e
i n c o r p o r a t i o n o f C - l e u c i n e more th a n 2 - f o l d , a s shown
i n T ab le 9» and had a s i m i l a r e f f e c t on C ^ - v a l i n e
i n c o r p o r a t i o n i n o t h e r e x p e r im e n ts . T h is amino a c id
m ix tu re was d e s ig n e d to y l t l d o p tim a l s y n t h e s i s o f
Q b
hem oglobin by whole c e l l s u s p e n s io n s o f r a b b i t r e t i c u l o
c y t e s . I n t h i s r e s p e c t , amino a c i d in c o r p o r a ti o n i n to
p r o t e i n i n th e c e l l - f r e e system re s e m b le s p r o t e i n s y n t h e s i s
by whole c e l l s ( 9 6 ). The a d d i t i o n o f an amino a c id
m ix tu re c o n ta in in g e a c h amino a c id a t a u n ifo rm c o n c e n tr a
t i o n (0 .2 5 pm oles p e r a s s a y ) was i n h i b i t o r y .
I n many c e l l - f r e e amino a c id in c o r p o r a ti o n sy stem s
(3 ^ , 1 0 0 ), th e r e a c t i o n c e a s e s a f t e r 15 to 20 m in u te s o f
in c u b a ti o n . I n v e s t i g a t i o n o f th e tim e c o u rse w ith
r e t i c u l o c y t e rib o so m es (co m p lete system ) shows an a lm o st
c o n s ta n t in c r e a s e i n in c o r p o r a ti o n f o r U -O m in u te s and
o c c a s i o n a l l y f o r 60 m in u te s . A k i n e t i c stu d y o f th e
i n c o r p o r a ti o n p ro c e s s shows t h a t th e i n i t i a l la b e li n g ta k e s
p la c e i n th e ribosom e p a r t i c l e s w hich a t t a i n a n e a r ly
c o n s ta n t s p e c i f i c a c t i v i t y in 10 m in u te s , and th e n th e r e
was a se co n d a ry r i s e i n l a b e l i n g o f p r o t e i n i n th e s o lu b le
f r a c t i o n o f th e r e a c t i o n m ix tu re w hich c o n tin u e s to r i s e
f o r a p p ro x im a te ly ^-0 m in u te s . T h is stu d y was made by
c e n t r i f u g a t i o n o f th e r e a c t i o n m ix tu r e , a f t e r in c u b a tio n ,
f o r 1 h o u r a t 105*000 x g , and w ashing rib o so m es and
s o lu b le p r o t e i n s s e p a r a t e l y f o r d e te r m in a tio n o f r a d i o
a c t i v i t y . R a d io a c tiv e rib o so m e s , l a b e le d i n in c u b a tio n s
o f s h o r t e r d u r a t i o n (10 to 15 m in u te s ) , c o u ld be i s o l a t e d
by c e n t r i f u g a t i o n and r e in c u b a te d w ith C ^ -a m in o a c i d s ,
showing t h a t th e C ^ -a m in o a c id i n th e ribosom e p r o t e i n i s
s u b s e q u e n tly r e l e a s e d I n th e form o f la b e le d s o lu b le
p r o t e i n . C hrom atography o f th e s o lu b le p r o t e i n s demon
s t r a t e d t h a t th e Cllf- l a b e l e d s o lu b le p r o t e i n s c o n s is te d
m ain ly o f la b e le d hem oglobin ( 9 9 ). A f a i l u r e to r e l e a s e
com pleted p r o t e i n s from th e m icrosom es i n o th e r c e l l - f r e e
system s m igh t a c c o u n t f o r th e c e s s a t i o n o f th e r e a c t i o n
a f t e r a s h o r t e r tim e .
F u r th e r in fo r m a tio n w hich i n d i c a t e s t h a t "amino
a c id i n c o r p o r a tio n " i n t h i s system i s synonymous w ith
s y n t h e s i s o f norm al c e l l p r o t e i n s was p ro v id e d by th e
r a t i o s o f th e am ounts o f C ^ - l a b e l e d l e u c i n e , i s o l e u c i n e ,
and v a li n e in c o r p o r a te d ( 9 9 ). These r a t i o s c o in c id e d
c l o s e l y w ith th e p r o p o r tio n s o f th e s e amino a c id s i n th e
t o t a l s o lu b le p r o t e i n s o f th e r a b b i t r e t i c u l o c y t e s . T h is
agreem ent i s f u r t h e r e v id e n c e t h a t th e s e p r o t e i n s , o f
w hich hem oglobin i s th e m ain c o n s t i t u e n t , a re b e in g
s y n th e s iz e d i n th e c e l l - f r e e sy stem . The d a ta a ls o p e rm it
a d i s t i n c t i o n to be made betw een th e " s t r u c t u r a l " p r o t e i n
o f th e ribosom e and th e p r o t e i n b e in g s y n th e s iz e d . In th e
r e t i c u l o c y t e s o lu b le p r o t e i n s , th e i s o l e u c in e c o n te n t was
o n ly 12$ o f th e amount o f le u c in e found i n th e p r o t e i n s .
S ince a p p ro x im a te ly 50$ o f th e r a d i o a c t i v i t y rem ained i n
th e m icrosom es and th e i s o l e u c in e and le u c in e c o n te n t o f
th e rib o s o m a l p r o t e i n a re n e a r l y th e same (1 0 1 ), in c o r p o
r a t i o n i n t o rib o so m a l p r o t e i n would have changed th e
86
r a t i o s . T h e r e f o r e , i t i s p ro b a b le t h a t th e r a d i o a c t i v i t y
i n th e rib o so m es l a r g e l y r e p r e s e n t s hem oglobin p r e c u r s o r s .
In g e n e r a l , t h i s would im ply t h a t th e r i b o n u c l e o p r o t e i n
s e rv e s a s a te m p la te f o r p r o t e i n s y n t h e s i s b u t n o t a s a
p r e c u r s o r o f th e p r o t e i n s y n th e s iz e d .
The r o l e o f t r a n s f e r r i b o n u c l e i c a c id
i n hem oglobin s y n th e s is
The s t i m u l a ti o n o f amino a c id in c o r p o r a ti o n i n t o
m icrosom al p r o t e i n by p o ly n u c le o tid e p r e p a r a t i o n s h as been
n o te d by Zam ecnik e t a l . (102) and W eiss e t a l . (6 7 ) .
More r e c e n t l y , R endi and Cam pbell (103) have r e p o r te d i n
c re a s e d amino a c id in c o r p o r a ti o n when e i t h e r s o lu b le o r
m icrosom al RNA was added to a p r e p a r a t i o n c o n ta in in g l i v e r
m icrosom es. In th e p r e s e n t s t u d i e s (10*4), i n c o r p o r a ti o n
o f Cll+- l e u c in e i n t o p r o t e i n u sin g rib o so m es from r a b b i t
r e t i c u l o c y t e s was s tim u la te d s p e c i f i c a l l y by t r a n s f e r RNA
(T able 1 0 ) .
E i t h e r t r a n s f e r RNA from g u in e a p ig l i v e r (th e
m ost p u r i f i e d RNA f r a c t i o n ) o r s o lu b le r e t i c u l o c y t e RNA
gave com parable i n c r e a s e s i n C ^ - l e u c i n e i n c o r p o r a ti o n
in t o p r o t e i n when s i m i l a r l e v e l s (a s sa y e d by C ^ - l e u c i n e -
RNA fo rm a tio n ) were u se d . Maximal e f f e c t s were o b ta in e d
w ith 25 to *+0 jig o f t r a n s f e r RNA. These r e s u l t s depended
on th e p r e p a r a t i o n o f th e s o lu b le enzyme f r a c t i o n f r e e o f
t r a n s f e r RNA. The i n c o r p o r a ti o n shown i n th e ab sen ce o f
87
TABLE 10
EFFECT OF RIBONUCLEIC ACID O N Cll+-LEUCINE
INCORPORATION INTO PROTEIN
S p e c if ic a c t i v i t y
RNA added----------------------------------- ;------- ------
c .p .m ./m g o f
rib o so m es
N o n e i/ ........................................................... 6*+3
Transfer RNA2/ ................. 1591
Reticulocyte soluble RNA .... 16^0
Microsomal RNA ................. 76*+
Preincubated R N A ................. 68*+
Preincubated RNA + CTP ........ 939
Preincubated RNA + CTP +
Fraction AS-1 ............... 1258
-^The s ta n d a rd a s s a y c o n d itio n s (m inus RNA) were
u se d , w ith 7 mg o f ribo so m es from r a b b i t
r e t i c u l o c y t e s . When CTP was ad d ed , th e concen
t r a t i o n used was 0 .1 5 pmole p e r ml o f r e a c t i o n
m ix tu r e . A pproxim ately 100 pg o f F r a c t i o n AS-1
was used where i n d i c a t e d .
2/
- T r a n s f e r RNA (50 pg p e r a s s a y ) , r e t i c u l o c y t e
s o lu b le RNA (100 p g ) , m icrosom al RNA from
r e t i c u l o c y t e ribosom es (100 p g ), o r p r e i n
c u b ated RNA (100 pg) were used where i n d i c a t e d .
The p r e p a r a t i o n o f th e s e ty p e s o f RNA was
d e s c r ib e d i n th e s e c t i o n on m eth ods.
88
added RNA (T ab le 10) may be due to th e p re s e n c e o f a sm a ll
amount o f t r a n s f e r RNA i n th e rib o s o m e s . T h is may a l s o
a c c o u n t f o r th e s l i g h t s t i m u l a t i o n o f i n c o r p o r a t i o n shown
when m icrosom al RNA ( p re p a re d from r e t i c u l o c y t e rib o so m es
by th e u s u a l p h e n o l m ethod) was a d d ed . T u rn ip y e llo w
m osaic v i r u s RNA and com m ercial y e a s t RNA gave no
s t i m u l a t i o n o f i n c o r p o r a t i o n .
The f u n c t i o n a l i n t e g r i t y o f th e RNA a s amino a c i d -
a c c e p to r was needed f o r s t i m u l a t i o n o f th e i n c o r p o r a t i o n
o f amino a c i d s i n t o p r o t e i n . P re in c u b a te d RNA ( 8 3 ) , w hich
h as l o s t te r m in a l n u c l e o t i d e s , d id n o t s t i m u l a te C ^ -
le u c in e i n c o r p o r a t i o n i n t o p r o t e i n i n th e s ta n d a r d system
(T ab le 1 0 ) . T h is RNA does n o t form amino acid-RNA u n le s s
CTP and F r a c t i o n AS-1, w hich r e p l a c e s t e r m in a l n u c le o
t i d e s , a re ad d ed . S i m i l a r l y , th e s t i m u l a t i o n o f in c o r p o
r a t i o n i n t o p r o t e i n w ith p re in c u b a te d RNA depended on th e
a d d i t i o n o f F r a c t i o n AS-1 and CTP. T h e r e f o r e , th e r o l e
o f th e RNA i n t h i s s t i m u l a t i o n a p p e a re d to be due to i t s
a b i l i t y to form amino acid-RNA compounds. When F r a c t i o n
AS-1 and CTP were added to th e com plete system c o n ta in in g
t r a n s f e r RNA, no e f f e c t o r a s l i g h t i n h i b i t i o n was
o b s e rv e d . T h is f in d in g i n d i c a t e s t h a t th e rem oval and
re p la c e m e n t o f t e r m in a l n u c l e o t i d e s i s p ro b a b ly n o t
in v o lv e d d i r e c t l y i n hem oglobin s y n t h e s i s i n th e c e l l - f r e e
sy ste m .
89
The in c r e a s e d Cll+- l e u c in e in c o r p o r a ti o n i n t o
p r o t e i n upon a d d i t i o n o f RNA i n a la r g e number o f e x p e r i
m ents ran g e d from 15 t o *+0 tim e s th e amount o f C ^ -
leucine-R N A w hich was form ed by t h a t amount o f RNA i n th e
s ta n d a rd a s s a y o f amino acid-RNA f o rm a tio n . A ,,c a t a l y t i c ,,
r o l e was t h e r e f o r e i n d ic a t e d f o r RNA i n th e t r a n s f e r o f
amino a c i d s i n t o p r o t e i n . T h is s u g g e s tio n was t e s t e d
e x p e r im e n ta lly by p r e in c u b a tin g th e s o lu b le com ponents
(minus rib o s o m e s ), in c lu d in g e i t h e r t r a n s f e r RNA o r
1 L l
r e t i c u l o c y t e RNA, w ith C - l e u c in e f o r 5 m in u te s . A f te r
1 O
t h i s p e rio d o f p r e in c u b a tio n , a 1 0 0 - fo ld e x c e s s o f -
le u c in e was added and th e n th e rib o so m es were im m e d ia te ly
added and th e in c u b a tio n c o n tin u e d f o r th e u s u a l *+0
m in u te s . The i n c o r p o r a ti o n o f th e le u c in e i n to
p r o t e i n was d e te rm in e d on t h i s sam ple. A d u p l ic a t e a s s a y
(w ith o u t a d d it i o n o f rib o so m es) was im m e d ia te ly p r e
c i p i t a t e d w ith c o ld t r i c h l o r o a c e t i c a c id a f t e r a d d i t i o n
o f th e C ^ -^-leu cin e, and a d e te r m in a tio n was made o f th e
amount o f C ^ -le u c in e -R N A form ed i n th e p r e i n c u b a t i o n .
The c o n d itio n s o f th e p r e in c u b a tio n were such t h a t m axim al
■ ] L
i n c o r p o r a tio n o f C - l e u c in e i n t o RNA to o k p la c e i n th e
5 m inute i n c u b a tio n . To a n o th e r d u p l ic a t e a s s a y , th e
ribosom es were added to th e p r e in c u b a tio n m ix tu re w ith o u t
th e a d d i t i o n o f C ^ - l e u c i n e and th e in c u b a tio n c o n tin u e d
a s b e fo re and r a d i o a c t i v i t y i n p r o t e i n d e te rm in e d .
90
The r e s u l t s o f th e ty pe o f e x p e rim e n t j u s t
d e s c r ib e d , shown i n T ab le 1 1 , have i n d i c a t e d t h a t a l l th e
C ^ - l e u c i n e in c o r p o r a te d i n t o p r o t e i n i n t h i s system
p a s s e s th ro u g h amino acid-RNA compounds. The amount o f
C l^-leucine-R N A form ed i n th e p r e in c u b a tio n was a lm o st
q u a n t i t a t i v e l y t r a n s f e r r e d to p r o t e i n (89 to 96%) i n th e
r e g u l a r in c u b a tio n system under c o n d itio n s where th e
e x c e ss C - ^ - l e u c i n e p r e s e n t would e x clu d e th e fo r m a tio n o f
more C ^ -le u c in e -R N A . I f no C ^ - l e u c i n e were added a f t e r
th e p r e in c u b a tio n , th e r a t i o o f c o u n ts found i n p r o t e i n to
th e amount o f C -^-leucine-R N A form ed was 2 0 .5 u sin g
t r a n s f e r RNA and 1 6 .5 u sin g r e t i c u l o c y t e RNA. The same
s t i m u l a ti o n o f ClLf- l e u c in e in c o r p o r a ti o n i n t o p r o t e i n h as
been o b ta in e d i n o th e r e x p e rim e n ts w ith o n ly h a l f th e
amount o f RNA used i n T able 11 , so t h a t a r a t i o o f *+0 h as
a ls o been o b ta in e d .
Some p r e lim in a r y s t u d i e s , u sin g th e method o f
p r e in c u b a tio n o f s o lu b le components to form C ^ -a m in o
acid-RNA (T able 1 1 ), have shown t h a t th e e x te n t o f
t r a n s f e r o f th e r a d i o a c t i v e amino a c id from RNA to p r o t e i n
was d ep en d en t on th e p re s e n c e o f a m ix tu re o f th e o th e r
amino a c id s and a c t i v a t i n g enzym es. T h is f in d in g
i n d i c a t e s t h a t th e fo rm a tio n o f a m ix tu re o f th e v a r io u s
amino acid-RNA compounds i s p ro b a b ly r e q u i r e d f o r th e
t r a n s f e r o f th e s in g le C -am ino a c id i n t o p r o t e i n .
91
TABLE 11
TRANSFER OF AMINO ACIDS FROM RNA TO PROTEIN
RNA added i n
p r e in c u b a tio n
1 /
A d d itio n s a f t e r
5 m in u te s
i T S i * :
l e u c i n e -
RNA
T o ta l
r * r* Ttf
E x cess
C l2-
l e u c in e
R ibo
somes
2 /
i n
p r o t e i n
y
T r a n s f e r RNA
+ —
!+30 0
T r a n s f e r RNA
+ +
375
T r a n s f e r RNA
- +
981+8
R e tic u lo c y te
RNA
+
..
678 0
R e tic u lo c y te
RNA
+
668
R e tic u lo c y te
RNA -
+ - 1 1 2 0 0
“ The s ta n d a rd a s s a y c o n d itio n s (m inus rib o so m e s , GTP,
and p o ta s siu m c h lo r i d e ) were used i n th e p r e in c u b a tio n
w ith 100 pg o f t r a n s f e r RNA o r 300 pg o f r e t i c u l o c y t e
RNA where i n d i c a t e d .
2/
- A s o l u t i o n o f rib o so m es c o n ta in in g th e u s u a l am ounts o f
GTP and p o ta ssiu m c h lo r id e used i n th e com plete system
was added im m e d ia te ly a f t e r th e a d d i t i o n o f C l2 - le u c in e
when b o th a d d it i o n s were made a f t e r 5 m in u te s o f p r e
i n c u b a tio n .
^ T h e t o t a l c o u n ts p e r m inute i n p r o t e i n a re g iv e n a s
th o s e d ep en d en t on th e a d d i t i o n o f RNA. D u p lic a te s
were ru n i n e ac h c a te g o ry w ith o u t th e a d d i t i o n o f RNA.
92
C-^-Threonine-RNA, formed in the preincubation system by
the use of Fraction AS-1 (100-fold purified threonine-
activating enzyme) in place of the usual Reticulocyte
Fraction AS *4-0-70, transferred only a small fraction of
the C^-threonine to protein when excess C^-threonine
and ribosomes were added. The extent of transfer was in
creased significantly if the amino acid mixture and the
usual reticulocyte enzyme fraction (containing a mixture
of activating enzymes and unknown enzymic components)
were added with the ribosomes.
In the earliest studies of amino acid-RNA
formation by Hoagland et al. (60), it was found that
transfer RNA labeled with amino acids in the whole pH 5
fraction would transfer the amino acids to microsomal
protein. This finding has been confirmed in this labora
tory in studies by Bishop et al. (105) using transfer RNA
labeled with C^-amino acid by activating enzymes and ATP,
and isolated by the phenol method. Incubation of this
material with ribosomes, 'magnesium ion, GTP, and an
enzymatic component of the soluble cell fraction resulted
in a rapid and irreversible transfer of the amino acid to
ribosomal protein.
V. DISCUSSION
The essential task before all investigators in the
field of protein synthesis is to discover how the cell puts
together in peptide linkage twenty amino acids in the
observed variety of specific, genetically determined
sequences. The variety of approaches to the problem is
rapidly increasing as the once bewilderingly complex
systems are beginning to be replaced by actual sequential
chemical reactions as based on experimental observation.
The demonstration that all of the common amino
acids can be activated in a single tissue, as measured by
PP^ exchange, supports the hypothesis that this reaction
may be the first step in protein synthesis (36). The
activation of an amino acid by a different mechanism,
however, has also been reported (106). Activating enzymes
for the amino acids studied here have very similar
properties, as evidenced by parallel loss in activity on
storage, pH optimum, inhibition by p-chloromercuribenzoate,
protection by GSH, and affinity for substrates. The in
creased stability of activating enzymes in the presence of
GSH, and the partial protection against inhibition by
p-chloromercuribenzoate observed when substrate was
present, indicates that a mechanism of action involving
93
essential thiol groups may be operative for most
activating enzymes.
The tyrosine- and threonine-activating enzymes
differed from others studied as they were much more stable
to storage and E-chloromercuribenzoate effected less
Inhibition. These enzymes also had a higher solubility in
ammonium sulfate and potassium phosphate. The basis for
this difference is not known, but may be of importance in
the understanding of the specificity of the activating
i
e n z y m e s i n t h e i r r e a c t i o n w i t h a m in o a c i d - s p e c i f i c
f r a c t i o n s o f t r a n s f e r RNA ( 8 5 ) .
Purification and characterization of single amino
acid activating enzymes has proceeded from bacterial,
avian, and mammalian tissues. It is a likely possibility
that twenty separate enzymes may take their place as tools
for biochemists. There seems to be no further doubt that
activating enzymes exist for all the amino acids (1 +3-L f5).
The low level of activity toward certain amino acids, and
the reports in which a complete complement of activating
enzymes could not be demonstrated, can best be explained
by assuming that the enzyme proteins, or a link between
these proteins and a key prosthetic group, vary in their
lability to the procedures used to isolate them. For
this reason, the extent of activation of various amino
acids does not reflect the composition of the protein of
the tissue from which the enzymes are derived.
There are still a number of interesting questions
relating to amino acid activation which are being pursued.
One of these concerns the role of the activating enzymes
in the determination of specificity of the finished
protein which they presumably help to synthesize. The
enzymes efficiently reject the D-isomers of the amino
acids and there is no evidence for competition between
different L-amino acids for the same site. Observations
by Sharon and Lipmann (107) and by Schweet and Allen (50)
have shown, however, that certain unnatural amino acid
analogs are activated by these enzymes and these in
general are the ones which ultimately appear in protein.
Thus, it would seem that there is considerable enzymatic
selectivity in this first step in protein synthesis.
I-n this discussion, three important aspects should
be re-emphasized concerning the role of RNA in protein
synthesis: first, there is general agreement that amino
acid activation, i.e., the formation of the enzyme bound
acyl-adenylate compounds, is an enzymatic reaction
entirely independent of RNA. This statement is based on
the fact that ribonuclease does not affect the activation
of amino acids by crude or purified activating enzyme
preparations, and that the more highly purified activating
enzymes do not contain measurable RNA and are fully active
96
in the absence of RNA. Second, it appears highly probable
that each amino acid is activated by its own single
specific enzyme. Third, the activated amino acyl-
adenylate intermediate is very firmly bound to its specific
enzyme and dissociates to only a very small extent in the
absence of an acceptor for the amino acid. The activating
enzyme and its bound amino acyl-adenylate can in effect
be thought of as acting as a single unit.
The studies presented here have shown the need for
a specific activating enzyme to catalyze the formation of
the corresponding amino acid-RNA. Purification of the
threonine-activating enzyme indicated that the transfer of
amino acid to RNA is probably mediated directly by the
activating enzyme without the involvement of other
enzymes. Very low concentrations of the 100-fold purified
enzyme catalyze the rapid incorporation of C^-threonine
into RNA. This conclusion has also been strongly sup
ported by the work of Berg and Ofengand (65) who have
shown that during the course of a 100-fold purification of
valine-activating enzyme there was a parellel increase in
the ability of fractions to catalyze activation, and
transfer of the amino acid to RNA. There has also been
recent evidence reported that synthetic amino acyl-
adenylates may serve as sources of amino acid on transfer
RNA provided they are first bound to activating
97
enzymes (108). The possibility that enzymes in addition
to the activating enzyme may be acting in amino acid-RNA
formation has not been completely excluded. Further
enzyme purification will be needed to settle this
question, although no evidence in favor of this possi
bility has been found in these studies.
The particular RNA which has suddenly taken the
limelight on the protein synthesis stage appears to have
generally similar properties in both bacterial and animal
tissues. It is best isolated from the soluble fraction
of tissues by treatment with phenol, followed by alcohol
precipitation and dialysis— a method first used by
Gierer and Schramm (8l) to isolate competent tobacco
mosaic virus RNA. Partial purification of an RNA fraction
active for amino acid incorporation has indicated the
occurrence of a unique type of RNA with a specific func
tion. This RNA has been termed "transfer RNA" to denote
its function in transferring activated amino acids to
specific sites in the ribosome where they are then linked
to adjacent amino acids.
The molecular weight of "soluble" RNA (nonribo-
somal RNA) based on ultracentrifuge studies has been
reported as 20,000 to 50,000 (60, 109). Direct analysis
of the amount of adenosine released on alkaline hydrolysis
of guinea pig liver transfer RNA permitted the estimation
of the molecular weight as 30,000 (73). The real value
may be lower since impurities and losses of adenosine all
tend to make the estimate high. Somewhat higher values of
around 50,000 have been indicated for the E. coli transfer
RNA preparation (110). An estimate based on the average
amount of amino acid incorporated into transfer RNA in the
present studies gives a molecular weight value of around
28,000, and the amount of C-^-ATP incorporated into pre
incubated pH 5 RNA was equivalent to a molecular weight
of 2*f,500 for transfer RNA.
Studies on the base composition of transfer RNA
are only in preliminary stages, but even these early
results add support to the concept that this RNA is a
unique, specific type of cellular RNA. The discovery by
Cohn and Volkin (111, 112) and Allen and coworkers (113»
ll^f) of “pseudouridine" (5 ribosyl uracil) has led to an
exploration of its location in definable cellular RNA
fractions. Dunn (115) has subsequently noted that this
nucleoside is considerably more abundant in soluble RNA
than in microsomes. He also observed that there was a
relative excess of methylated derivatives of the bases in
soluble RNA. The metabolism of transfer RNA also is
unique, based on studies of turnover (86, 116, 117) and
the terminal addition of nucleotides (90, 118-122).
99
Early observations of amino acid-RNA Interaction,
which have been confirmed and extended in the studies
presented here, indicated that amino acids were additively
and non-compe111ively attached to transfer RNA. This
finding clearly established the fact that separate sites
were available for each amino acid. Zachau et al. (8 9)
then demonstrated directly that the amino acids were
esterifled on the terminal adenosine of the RNA. This was
done by treatment of leucine-labeled transfer RNA with
pancreatic ribonuclease which resulted in a quantitative
yield of leucyl-adenosine. It has been shown in these
studies and by Hecht et al. ( 8 3, 122) that the terminal
configuration of nucleotides was necessary for amino acid
attachment. Preiss et al. (110) and Hecht et al. (122)
also showed that periodate treatment of the RNA resulted
in loss of ability to accept amino acids and that amino
acids protected against the action of periodate. Periodate
would be expected to oxidize any free 2', 31 hydroxyl
groups. This experiment was carried one step further
(110) by showing that amino acids could protect only
their own sites of attachment to RNA from periodate
oxidation.
The similarity in amounts of adenosine released
by alkaline hydrolysis of RNA and amino acid incorporated
confirms the more direct evidence cited above for the
linkage of amino acids to the terminal adenosine of
transfer RNA. The requirement for terminal nucleotides
for amino acid incorporation was directly shown here with
preincubated RNA. In these studies, an activating enzyme
fraction which did not catalyze the replacement of
terminal nucleotides into RNA was unable to catalyze in
corporation of C^-leucine, or C^-isoleucine, into pre
incubated RNA. Incorporation was restored with the
iL
addition of an enzyme fraction which catalyzed C -ATP
incorporation into this RNA. In contrast, amino acid
incorporation into transfer RNA was maximal when activat
ing enzymes free of C-^-ATP incorporating activity were
used. This finding indicated that transfer RNA, as
isolated, contained almost all of the terminal AMP
required for amino acid incorporation and, therefore,
needed no replacement of terminal groups.
It should perhaps be stated explicitly that the
terminal AMP grouping of the RNA to which the amino acid
becomes attached is not derived from the AMP moiety of the
amino acyl-adenylate which furnishes the amino acid.
This was considered a possibility at one time but has been
clearly ruled out by the facts that 1) terminal AMP
attachment is clearly catalyzed by enzymes other than
those which activate amino acids; and 2) in crude systems
containing both activities terminal AMP attachment is not
101
dependent on amino acids (122).
It was apparent in the early studies of the
chemical behavior of the amino acid-RHA link that a
phosphoanhydride type of bond was precluded. The bond was
quite stable at pH's below neutrality even at high
temperature, and in alkali it hydrolyzed at a rate con
siderably slower than amino acyl phosphoanhydrides. It
was also much less reactive with hydroxylamine and
ammonia. The periodate inactivation studies mentioned
previously were also strongly indicative of an ester bond
between amino acid carboxyl and ribose 2' or 3' hydroxyl
group, a configuration which has now been established as
correct in both mammalian (8 9) and bacterial (110) systems
by direct removal of the terminal adenosine-amino acid
ester by ribonuclease.
This particular type of linkage is of special
interest because it is in equilibrium with the AMP-
pyrophosphoryl bond of ATP. This equilibrium has been
demonstrated by the reversibility of amino acid-RNA
formation shown in the results presented here and by the
incorporation of Cll+-AMP into ATP when isolated threonine-
RNA was cleaved in the absence of ATP (8^). An
equilibrium constant for the reaction ATP + RNA +
threonine k threonine-RNA + AMP + PP^ has been
determined from both directions as 0.37 at pH 7.0 by
102
Leahy (u n p u b lish ed ex p erim en ts) i n our la b o r a to r y .
S i m i la r ly , Berg and coworkers (5*0 found a valu e o f 0 .3 2
f o r valine-RNA fo rm a tio n . A sim ple e s t e r bond would
c e r t a i n l y n o t be e x p ected to have such a high energy con
t e n t and i t i s t h e r e f o r e assumed t h a t th e presence o f the
a d ja c e n t hydroxyl group on th e r ib o s e c o n fe rs s p e c ia l
r e a c t i v i t y p r o p e r t i e s to th e lin k a g e .
When it was found that amino acids were attached
to specific terminal sites on transfer RNA, it became
apparent that transfer RNA must consist of a number of
distinct species. Much work has since been directed
toward attempts to fractionate the soluble RNA, both to
separate the transfer RNA's from one another and to
separate them from contaminating unrelated RNA. Smith
et al. (85) have effected some preliminary fractionation
of transfer RNA's specific as acceptors for different
amino acids by the use of substituted cellulose columns.
Another approach to transfer RNA fractionation has been
the use of countercurrent distribution as reported by
Holley and Merrill (123). These workers have obtained
partial separation of the transfer RNA which accepts
alanine by this means. Because of the relatively high
molecular weight of the specific transfer RNA molecules
and the probability that the molecular weight distribution
is narrow, fractionation based on size, solubility,
electrical charge, or difference In base content Is not
considered as a simple task. For this reason, several
laboratories are now attempting to isolate a particular
transfer RNA by making use of the amino acid it specifi
cally binds, or by making use of the fact that the amino
acid protects its particular RNA from oxidation of the 2*
and 3' hydroxyl groups of the terminal ribose residue by
periodate.
Establishment of the fact that amino acid-RNA
compounds are true intermediates in protein synthesis,
requires that they fulfill kinetic specifications for an
intermediate in vivo and the demonstration in vitro that
they may serve directly as the source of amino acid in
specific newly synthesized protein molecules. Hoagland
et al. (60) slowed the rate of labeling of ribosomal
protein in ascites tumor cells by incubation at a reduced
temperature. Following the exposure of the cells to a
C^-amino acid, the radioactivity appeared initially in
the soluble RNA fraction, rising rapidly to a plateau
value. At a slower rate the amino acid found its way into
particle protein and even more slowly but progressively
the label finally appeared in soluble protein. Con
siderably more detailed studies on the in vivo relation
ship of these fractions have been carried out by Lacks
and Gros (121 +). These investigators have confirmed the
mammalian results in an E. coli system and have elegantly
established two further points. First, the rate of
attachment of amino acids to transfer RNA was found to be
a function of the rate of protein synthesis. Inhibition
of protein synthesis slows the rate of equilibration of a
C^-amino acid with the transfer RNA pool but does not
alter the final equilibrium level. Amino acids of which
the cells have been deprived equilibrate much more
rapidly with the pool. Observations such as these suggest
that the availability of sites on transfer RNA is a
direct function of the rate at which sites are made
available by subsequent steps in protein synthesis.
Second, the removal of the inhibition of protein synthesis
results in a transfer of the labeled amino acid from the
RNA pool to protein.
Thus, it is reasonable to conclude that the
kinetic behavior of transfer RNA in vivo suggests that it
is an intermediate in protein synthesis. The cell-free
system studied here has brought to light some further
support of the role of transfer RNA-amino acid as an
intermediate. The maximal incorporation of free amino
acids into hemoglobin using ribosomes from rabbit
reticulocytes has been shown to take place only in the
presence of transfer RNA, intact with respect to its
ability to accept amino acids.
105
Transfer RNA labeled with amino acid in the
absence of ribosomes transfers the amino acid to ribosome
1 L
protein. The C -amino acid, in its transit to protein,
1 9
did not pass through a free state since its C- 1 - -homolog
was present in excess. If the C^-amino acid were in
corporated into RNA in the absence of the usual complement
of unlabeled amino acids, the resulting transfer to
protein was much less effective in donating its amino acid
to ribosomal protein. This would appear to mean that the
other unlabeled amino acids usually bound to transfer RNA
in the incubation are required for the incorporation of
the single labeled amino acid and most likely are in
corporated into protein with it.
Further experimentation may show that there are
alternate pathways of protein synthesis. Certainly the
results on studies of protein synthesis in cell fractions
other than ribosomes and microsomes point to this pos
sibility. The experiments reported in this dissertation,
however, do not justify any conclusion other than the
participation of amino acid-RNA formation as a true
intermediate step in protein synthesis.
The occurrence of these reactions between RNA and
amino acids has tempted many to construct theories of
template function upon these observations. One of the
most interesting theories has been the "adaptor hypothesisl*
106
Hoagland (125) has stated this explicitly as followsj
Amino acids, before entering the ribonucleo-
protein particles first react with small polynucleo
tide molecules. These adaptor molecules accompany the
amino acids into the particles and are responsible
for properly locating them on the particle RNA. This
is accomplished by pairing of the adaptors' bases with
complementary base sequences on the particle RNA.
Having completed their mission, the adaptors then
return to the soluble milieu.
Such a concept was arrived at independently by
Crick (126) on theoretical grounds. Experimental observa
tions are already in good agreement with the broad idea
of an adaptor serving as an intermediate, and for the
first time, a direct experimental attack on the coding
problem may be possible. It is of fundamental importance
to determine the minimal structural requirements of
transfer RNA underlying the specificity with which it
reacts with amino acids. Presumably the secret lies in
the sequence of bases in some part of the molecule, and
with the refinement of techniques for studying nucleic
acids, there is hope of finding the answer to one of
nature's most baffling mysteries— the mechanism by which
cells code amino acids in specific sequence and thus
express their genetic information. Whether the concepts
and theories concerning protein synthesis discussed here
represent a correct picture of the actual process remains
to be seen. What is much more important is that all
phases of the problem are now open to immediate
experimental attack
VI. SUMMARY
A study of amino acid activation in guinea pig
liver has shown that all of the common amino acids were
activated as measured by pyrophosphate exchange. The
inactivation of most of these enzymatic activities was
partially prevented by the use of reduced glutathione in
storage and incubation of the enzymes. A fractionation
scheme was developed for obtaining separation of active
ribonucleic acid and various activating enzymes.
Threonine-activating enzyme was purified 100-fold compared
to its specific activity in the pH 5 enzyme fraction.
The various activating enzymes studied evidenced very
similar properties.
The requirement for activation of a given amino
acid in order to obtain the formation of that amino acid-
RNA compound was demonstrated with specific activating
enzyme fractions. The relationship of the amino acid
activation reaction and amino acid-ribonucleic acid
formation was studied from several aspects. The incorpo
ration of thirteen C-^-amino acids into ribonucleic acid
was demonstrated with the pH 5 enzyme fraction, each
showing dependence on adenosine triphosphate as an
energy source.
108
109
Ribonucleic acid from various cell fractions was
tested for the ability to incorporate C^-amino acids. A
fraction which was purified 2-fold over the pH 5 ribo
nucleic acid indicated that a unique type of cellular
ribonucleic acid participates in the reaction. This type
of ribonucleic acid has been termed "transfer ribonucleic
acid" to denote its function in transferring activated
amino acids to specific sites in the ribosome where they
are then linked to adjacent amino acids.
Using activating enzymes from guinea pig liver,
ribonucleic acid prepared from several different species
was active for amino acid incorporation. Ribonucleic
acid from Escherichia coli. however, showed a slow rate of
incorporation with liver activating enzymes. Some
physical and chemical characteristics of transfer ribo
nucleic acid were studied and have been described.
Enzymes which catalyze the addition of terminal
adenosine-5'-monophosphate to ribonucleic acid have been
separated in function from activating enzymes. The former
enzymes were not required for incorporation of amino acids
into transfer ribonucleic acid. When ribonucleic acid
was incubated to remove terminal nucleotides, activating
enzymes and the enzymes which replace terminal nucleotides
were required for amino acid incorporation. These results
and others have led to the concept that a single amino
110
acid is attached to the terminal adenosine of a specific
polynucleotide chain.
The investigation of the enzymatic formation of
amino acid-ribonucleic acid compounds as intermediate re
actions in protein synthesis was carried out with a cell-
free system from rabbit reticulocytes. This system
incorporates amino acids into hemoglobin which could be
isolated and identified. Requirements of this system for
maximal incorporation were studied and briefly reported
here. The most interesting requirement, with respect to
the present research, was for transfer RNA, intact in its
function as an amino acid acceptor. Preincubated ribo
nucleic acid did not stimulate hemoglobin synthesis unless
terminal nucleotides were replaced, so that amino acid-
ribonucleic acid formation could take place. The amount
of transfer ribonucleic acid required for maximal stimula
tion of incorporation was so small that a "catalytic'1 role
for transfer RNA in protein synthesis has been suggested.
A labeled amino acid-ribonucleic acid prepared by incuba
tion of the components of the cell-free system without
ribosomes, was shown to transfer its amino acid to protein
when ribosomes were added. This process appeared to depend
on the presence of a complement of unlabeled amino acid-
RNA compounds.
Ill
The future significance of the reaction of amino
acid-ribonucleic acid formation studied here seems to be
in the further unraveling of the mysteries of the
specialized process of synthesis of specific protein.
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S tr u c tu r e o f N u cleic Acids and T h e ir Role i n
P r o t e in S y n t h e s i s . Cambridge U n iv e r s ity P r e s s ,
Cambridge, 1 9 5 7 , 'p. 25.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Allen, Esther Haney (author)

Core Title Studies On The Enzymatic Formation Of Amino Acid - Ribonucleic Acid Compounds

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Saltman, Paul (committee chair), Mehl, John W. (committee member), Webb, John L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-78287

Unique identifier UC11358053

Identifier 6005469.pdf (filename),usctheses-c18-78287 (legacy record id)

Legacy Identifier 6005469.pdf

Dmrecord 78287

Document Type Dissertation

Rights Allen, Esther Haney

Type texts

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

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

Repository Name University of Southern California Digital Library

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