Vol. 63, No. 1,1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
N-ACETYLPHENYLALANYL-tRNA
SPECIFIC
H Y D R O L A S E IN Y E A S T
M . A . B a j o , M . C . M o r a t i l l a and C . F . I n s t i t u t o de E n z i m o l o g £ a del C . S . I . C . ,
Heredia
F a c u l t a d de M e d i c i n a
de la L l n i v e r s i d a d A u t b n o m a , M a d r i d - 3 4 , S p a i n Received
January
14,1975
SUMMARY: An e n z y m e h a s b e e n found and p a r t i a l l y p u r i f i e d f r o m y e a s t which h y d r o l y z e s N - a e e t y l p h e n y l a l a n y l - t R N A and i s d i f f e r e n t f r o m the w e l l characterized N-substituted aminoacyl-tRNA hydrolase. The enzyme splits the e s t e r bond b e t w e e n N - a e e t y l p h e n y l a l a n i n e and the 3 w - h y d r o x y l g r o u p of the t e r m i n a l n u c l e o t i d e in the tRNA. INTRODUCTION:
E n z y m e s h y d r o l y z i n g both p e p t i d y l - t R N A s and N - s u b s t i -
t u t e d a m i n 0 a c y l - t R N A s h a v e b e e n found in the c y t o p l a s m of both p r o k a r y o t i c (1-4) and e u k a r y o t i e (5-7) c e i l s . C o m m o n c h a r a c t e r i s t i c s
to t h e s e e n z y m e s
a r e t h e i r low l e v e l s of s p e c i f i c i t y with r e s p e c t to p a r t i c u l a r a m i n o a c i d s (1) o r p e p t i d y l r e s i d u e s (8) and t h e i r i n a b i l i t y to a t t a c k u n s u b s t i t u t e d a m i n o a c y l t R N A s {1-3). T h e p r o d u c t s of h y d r o l y s i s a r e the i n t a c t tRNA m o l e c u l e and the c o r r e s p o n d i n g N - s u b s t i t u t e d a m i n o a c i d o r p e p t i d y l r e s i d u e . An e n z y m e with t h e s e c h a r a c t e r i s t i c s
(N-substituted aminoacyl-tRNA hydrolase) was de-
t e c t e d (5) and c r y s t a l l i z e d (6) f r o m y e a s t e x t r a c t s .
We now r e p o r t on the
i d e n t i f i c a t i o n of a d i f f e r e n t e n z y m a t i c a c t i v i t y p r e s e n t in y e a s t which h y d r o l y z e s N - a e e t y l p h e n y l a l a n y l - t R N A m u c h m o r e e f f e c t i v e l y than o t h e r N - a c e t y l ated aminoacyl-tRNAs.
T h i s e n z y m e h a s b e e n p a r t i a l l y p u r i f i e d and s e p a -
r a t e d f r o m the p r e v i o u s i d e n t i f i e d h y d r o l a s e . M A T E R I A L S AND M E T H O D S :
Uniformly labeled
f
~
p4cJ
o b t a i n e d f r o m the R a d i o e h e m i e a l C e n t e r ( A m e r s h a m ,
L-amino
acids were
England). Crude yeast
tRNA w a s p u r c h a s e d f r o m B o e h r i n g e r . T h e c h a r g i n g of tRNA with r a d i o a c t i v e a m i n o a c i d s and the i s o l a t i o n of the l 1 4 0 J a m i n o a c y l - t R N A s w a s done
as described
Acetylation of the [14CJaminoacy1-tRNA was performed
u s i n g a c e t i c a n h y d r i d e (10). T h e e x t e n t of a e e t y l a t i o n w a s c h e c k e d by a l k a l i n 1 t r e a t m e n t of the a c e t y l a t e d [14C] a m i n o a c y l - t R N A f o l l o w e d by p a p e r c h r o Copyright © 1975 by Academic Press, Inc. All rights o f reproduction reserved.
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m a t o g r a p h y (9). In all c a s e s m o r e than 95% a c e t y l a t i o n was a c h i e v e d . P r e p a r a t i o n of the y e a s t e x t r a c t s .
The s t r a i n of y e a s t u s e d was the
hybrid S a c c h a r o m y c e s fraqilis x S a c e h a r o m y c e s dobzhanskii.
Cells were
grown as d e s c r i b e d e l s e w h e r e (9) c o l l e c t e d at the m i d d l e of l o g a r i t h m i c p h a s e by c e n t r i f u g a t i o n , w a s h e d t h r e e t i m e s with cold w a t e r and s t o r e d at -20 ° until u s e . One p a r t of y e a s t was g r o u n d in a cold m o r t a r with two p a r t s of s a n d and the p a s t e was s u s p e n d e d in 1-2 v o l u m e s of a cold b u f f e r
con-
taining 50 mM T r i s - H C l pH 7, 10 mM 2 - m e r c a p t o e t h a n o l , and 10 mM m a g n e s i u m a c e t a t e . The h o m o g e n a t e was c e n t r i f u g e d in the cold at 1 0 , 0 0 0 xg f o r 10 rain. and the p r e c i p i t a t e was d i s c a r d e d . The s u p e r n a t a n t was c e n t r i fuged at 105,000 xg in a S p i n e o u l t r a c e n t r i f u g e at 2 - 5 ° f o r one and a half h o u r . The s u p e r n a t a n t a f t e r this c e n t r i f u g a t i o n was u s e d as s o u r c e of e n z y m e and i s r e f e r r e d to as c r u d e y e a s t s o l u b l e f r a c t i o n . T h i s f r a c t i o n can be s t o r e d at -70 °- f o r s e v e r a l d a y s without s i g n i f i c a n t l o s s of a c t i v i t y . A s s a y f o r e n z y m a t i c a c t i v i t y . The a s s a y m i x t u r e (0.1 ml) c o n t a i n e d , u n l e s s o t h e r w i s e s t a t e d , the following c o m p o n e n t s : 50 mM I m i d a z o l - H C l pH 6 . 5 , 2 . 5 mM m a g n e s i u m a c e t a t e , 2 . 5 mM 2 - m e r c a p t o e t h a n o l , about 8 0 p g oftRNAchargedwithS0pmolesofN-acetyl
[14C]aminoaeids (specificactiv -
ity 140 )~C/pmol) and the a m o u n t of e n z y m e i n d i c a t e d in e a c h c a s e . The r e a c t i o n m i x t u r e s w e r e i n c u b a t e d at 30 ° f o r the i n d i c a t e d p e r i o d s of t i m e . At the end of the i n c u b a t i o n the r e a c t i o n was s t o p p e d by addition of 3 ml of 5 p e r cent cold t r i c h l o r o a c e t i c a c i d . The m i x t u r e s w e r e kept at 0 -° f o r 10 rain, and f i l t e r e d t h r o u g h g l a s s f i b e r p a p e r (Whatman G F / C ) ; the f i l t e r s w e r e w a s h e d t h r e e t i m e s with 3 ml e a c h t i m e of 5 p e r cent cold t r i c h l o r o a c e t i c acid and the r a d i o a c t i v i t y r e m a i n i n g in the f i l t e r s was c o u n t e d in a liquid scintillation spectrometer.
C o n t r o l s without added e n z y m e w e r e r u n i n p a r a l l e l
to e s t i m a t e the s p o n t a n e o u s h y d r o l y s i s of the s u b s t r a t e . P r o t e i n s w e r e d e t e r m i n e d by the m e t h o d of L o w r y et al (11). R E S U L T S AND D I S C U S S I O N : The h y d r o l y s i s of d i f f e r e n t N - s u b s t i t u t e d a m i n o a c y l - t R N A s by a c r u d e y e a s t s o l u b l e f r a c t i o n is shown in F i g 1.
The
r a t e s of h y d r o l y s i s of N - a c e t y l v a l y l - t R N A and N - a c e t y l l e u e y l - t R N A a r e s i m i l a r and c l o s e to t h o s e p r e v i o u s l y r e p o r t e d as being due to the y e a s t N - s u b s t i t u t e d a m i n o a e y l - t R N A h y d r o l a s e (6). H o w e v e r N - a c e t y l p h e n y l a l a n y l - t R N A is h y d r o l y z e d at m u c h h i g h e r r a t e s (10 to 50 t i m e s h i g h e r , depending on the
256
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r51~ !
E
b
e /
40~ (D
o_ v
]3
t¢
3o~ i
',
I0
2
S
?
20 03
a;
I¢
i -Jl
i
I0
5v ~
"o
,I
T
5
I0
15
z
t
,~
I f
?~
g
".; II\kl o ,,4
20
Time (min.) 20
40
60
80
Fraction number
Fig i. H y d r o l y s i s of N - a c e t y l [ 1 4 4 a m i n o a c y l - t R N A s by the y e a s t s o l u b l e f r a c t i o n . R e a c t i o n s w e r e c a r r i e d odt a s d e s c r i b e d in Mfiez~hgds using 10 511 of y e a s t s o l u b l e f r a c t i o n (35. r~ag p r o t e i n / m l ) . N - a c e t y l - [ C] - pJa~nylalanyltRNA ( ® - - e ) ; N - a c e t y l - [ ± ~ C ] v a l y l - t R N A ( o - - o ) ; N - a c e t y l - [ i 4 C ] l e u e y l - t R N A (x--x). Fig 2. F r a c t i o n a t i o n of the y e a s t h y d r o l a s e s on D E A E - S e p h a d e x A-50 c o l u m n . A c o l u m n of D E A E - S e p h a d e x A-50 (2.5 x 14 em) was e q u i l i b r a ted with 50 mM T r i s - H C l , pH 7 c o n t a i n i n g 10 mM 2 - m e r c a p t o e t h a n o l and 10 mM m a g n e s i u m a c e t a t e . The c o l u m n was loaded with 15 ml of c r u d e y e a s t s o l u b l e f r a c t i o n (33 mg p r o t e i n / m l ) , w a s h e d with 65 ml o f the e q u i l i b r a t i o n b u f f e r , and e l u t e d with a l i n e a r g r a d i e n t of 0.1 M - 0 . 4 M T r i s - H C 1 pH 7 c o n t a i n i n g 10 mM 2 - m e r e a p t o e t h a n o l and 10 mM m a g n e s i um a c e t a t e (total b u f f e r 500 ml). The flow r a t e was a d j u s t e d to 0 . 5 ml p e r m i n . and f r a c t i o n s of 6 ml w e r e c o l l e c t e d . A c t i v i t y in the fra~Lonst ~ was d e t e r m i n e d in 10 pl o r 50 pl a l i q u o t z . u s i n g e i t h e r N - a c e t y l - [ CJ p h e n y l a l a n y l - t R N A ( . - - e ) o r N - a c e t y l [14C] v a l y l - tRNA ( o - - o ) r e s p e c t i v e ly a s s u b s t r a t e s .
d i f f e r e n t e x t r a c t s ) . T h i s fact c o n t r a s t s with the a p p a r e n t low s p e c i f i c i t y of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e t o w a r d s p a r t i c u l a r a m i no a c i d s r e s i d u e s (6) and s u g g e s t e d the e x i s t e n c e in y e a s t of a d i f f e r e n t e n z y m a t i c a c t i v i t y acting p r e f e r e n t i a l l y on N - a c e t y l p h e n y l a l a n y l - t R N A .
In a
s e a r c h f o r this a c t i v i t y we u s e d the s a m e y e a s t s t r a i n and f o l l o w e d the f i r s t s t e p s of p u r i f i c a t i o n i d e n t i c a l to t h o s e d e s c r i b e d f o r the c r y s t a l l i z a t i o n of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e , which b a s i c a l l y involve
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TABLE
I
S e p a r a t i o n of y e a s t h y d r o l a s e s by f r a c t i o n a t i o n with a m m o n i m s u l f a t e
Volume {ml)
Proteins {mg/ml)
Activity p m o l e s / m g p r o t e i n / 1 0 min N-acetyl-
N-aeetyl-
~4C_]P h e - t R N A ~4CI V a l - t RNA
Crude soluble f r a c t i o n
15
33
150
4.5
D E A E - S e p h a d e x A - 5 0 , then 0-60~0 (NH4) 2 SO 4 p r e c i p i t a t e
5
7.7
14
34
60-80~o (NH4) 2 SO 4 p r e c i p i t a t e
5
4.8
1300
0
A c r u d e s o l u b l e f r a c t i o n was c h r o m a t o g r a p h e d on D E A E - S e p h a d e x A-50 as i n -
dicated in F i g 2. The pooled f r a c t i o n s {120 m l ) , e o n t a i n i n g both a c t i v i t i e s , w e r e f r a c t i o n a t e d by adding solid a m m o n i u m s u l f a t e to r e a c h the p e r c e n t a g e s of s a t u r a t i o n i n d i c a t e d . The p r e c i p i t a t e s w e r e d i s s o l v e d in 5 ml of a b u f f e r c o n taining 10 mM T r i s - H C 1 pH 7, 10 mM p o t a s i u m c h l o r i d e , 10 mM 2 - m e r c a p toethanol and 5 mM m a g n e s i u m a c e t a t e . A c t i v i t y was d e t e r m i n e d as s p e c i f i e d in M e t h o d s .
c h r o m a t o g r a p h y on D E A E - S e p h a d e x and f r a c t i o n a t i o n with a m m o n i u m s u l f a t e (6). The only d i f f e r e n c e was that at e a c h step during the p r o c e s s of p u r i f i c a t i o n , the f r a c t i o n s w e r e a n a l y z e d f o r h y d r o l y t i c a c t i v i t i e s using two s u b s t r a t e s : N - a c e t y l [ 1 4 c l v a l y l - t R N A ( t h e ° n e r ° u t i n e l y u s e d i n t h e p u r[i f i c a1 t i o n of the
N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e ) and N - a c e t y l [ 1 4 C ] p h e n y l a l a n y l ,
tRNA. F i g 2 shows the elution p r o f i l e of the a c t i v i t i e s of a c r u d e y e a s t p r e p a r a t i o n a f t e r c h r o m a t o g r a p h y on D E A E - S e p h a d e x . In line with the r e s u l t s in Fig 1, the total a c t i v i t y on N - a c e t y l p h e n y l a l a n y l - t R N A is m u c h g r e a t e r than that on N - a c e t y l v a l y l - t R N A . M o r e o v e r , the p e a k s of a c t i v i t i e s on t h e s e two s u b s t r a t e s a r e not c o i n c i d e n t . S o m e f r a c t i o n s (40 to 45) which e f f e c t i v e l y h y -
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%
//
20 :-'----'--~-~/
×
o
I0
20 Time
~0
k 40
L' 50
(rain.)
Fig 3. H y d r o l y s i s of N - a c e t y l - [ ][14C]phenglglanyl-tRNA with p u r i f i e d h y d r o l a s e and r e c h a r g i n g of the tRNA with [ l ~ C ] p h e n y l a l a n i n e . R e a c t i o n m i x t u r e s containing: 50 mM I m i d a z o l pH 6 . 5 , 1 m i V L 2 - m e r c a p t o e t h a n o l , 1 mM m a g n e s i u m a c e t a t e , 70 p m o l e s of N - a c e t y l -[±'~C] p h e n y l a l a n y l tRNA ( 2 1 , 0 0 0 c p m , s p . a c t . 140 ] a C / ) ~ m o l ) with ( I - - i ) o r without (zX__Zx) h y d r o l a s e p r e p a r a t i o n (20 jal of the 60-80% a m m o n i u m s u l f a t e f r a c t i o n of T a b l e I) w e r e i n c u b a t e d at 30 °-. A f t e r 20 rain i n c u b a t i o n , 50 j~l of a x t u r e c o n t a i n i n g 20 mM A T P , 100 mM m a g n e s i u m a c e t a t e , 0 . 1 5 mM C] p h e n y l a l a n i n e (Sp. a c t i v i t y 116 pO/Mmol) and 20 p l of c r u d e y e a s t s o l u b l e f r a c t i o n ( p r e v i o u s l y p a s s e d t h r o u g h S e p h a d e x G-25) as s o u r c e of p h e n y l a l a n y l - t R N A s y n t h e t a s e w e r e added and the i n c u b a t i o n was c o n t i n u e d . At the t i m e i n d i c a t e d acid i n s o l u b l e c o u n t s w e r e d e t e r m i n e d as i n d i c a t e d in M e t h o d s . C o n t r o l s with on equal a m o u n t ofn~teacylated y e a s t tRNA ( 2 . 6 A,~r_,~ units) ( o - - o ) i n s t e a d of N - a c e t y l [~ "C] p h e n y l a l a n y l /,ou rLm . . .
~
t R N A w e r e run in parallel in reactlons a m o u n t of hydrolase preparation.
mixtures containing
the s a m e
d r o l y z e N - a c e t y l p h e n y l a l a n y l - t R N A have v e r y low o r no d e t e c t a b l e a c t i v i t y on N - a c e t y l v a l y l - t R N A . T h e s e r e s u l t s c o n t r a s t with the a p p a r e n t equal e f f e c t i v i t y of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e to a t t a c k e i t h e r of t h e s e two s u b s t r a t e s (6). F u r t h e r s e p a r a t i o n of the e n z y m e acting p r e f e r e n t i a l l y on N - a c e t y l p h e n y l a l a n y l - t R N A f r o m the h y d r o l a s e p r e v i o u s l y r e p o r t e d (6) can be a c h i e v e d by f r a c t i o n a t i o n of the p o o l e d f r a c t i o n s f r o m the D E A E - S e p h a d e x c h r o m a t o g r a p h y , c o n t a i n i n g both a c t i v i t i e s , with a m m o n i u m s u l f a t e .
The r e s u l t s in
T a b l e I show that as r e p o r t e d (6) the N - s u b s t i t u t e d a m i n o a e y l - t R N A
259
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lase p r e c i p i t a t e s with 60% s a t u r a t i o n of ammonium sul fat e. In the 60-80% ammonium sulfate p r e c i p i t a t e t h e r e is the hydrolytic activity v e r y active on N-acetylphenylalanyl-tRNA but which does not hydrol yze N - a c e t y l v a l y l tRNA to any a p p r e c i a b l e extent. P r e l i m i n a r y e x p e r i m e n t s have shown that this f r a c t i o n n e i t h e r h y d r o l y z e s phenylalanyl-tRNA nor the N-aoetylated d e r i v a t i v e s of leucyl_ and methionyl-tRNAs. A study on the speci fi ci t y of this enzyme is now in p r o g r e s s . The r e a c t i o n c a t a l yz e d by this enzym e involves the cleavage of the e s t e r bond between N-acetylphenylalanine and the 31-hydroxyl group of the 3 f t e r m i n a l nucleotide in the tRNA. This has been d e m o n s t r a t e d by r e c h a r g ing the deacylated N-acetylphenylalanyl-tRNA, by the action of the hydrolase, with r_[14Cj phenylalanine (Fig. 3). As it is shown in the F i g u r e the tRNA could be co m pl et el y r e c h a r g e d . The e x c c e s of charging o v e r the input counts is p r e s u m a b l y due to the f r e e tRNA s pe ci fi c for phenylalanine contaminating the p r e p a r a t i o n s of N-acetylphenylalanyl-tRNA. The r ol e of this enzym e in the cell, although still unknown, might be r e l a t e d in someway with the initiation of protein b i o s y n t h e s i s . In this respect it is imp o r tan t to note that N-aeetylphenylalanyl-tRNA acts as analogue of the N - f o r m y l m e t h i o n y l - t R N A F and is r e c o g n i z e d by the p r o k a r y o t i c i n i t i a tim f a c t o r IF 2 or its equivalent in e u k a r y o t i c cells (12 ,J 13). ACKNOWLEDGEMENT S t This work was supported by a grant f r o m the Fondo National pa r a el D e s a r r o l l o de la Investigacibn Cient{fica. REFERENCES 1.
Cuzin, F . , K r e t c h m e r , N . , G r e e n b e r g , R . E . , Hurwitz, R. and Chapeville, F. ( 1 9 6 7 ) P r o c . NatL Acad. S e i . LISA. 5_8, 2079-2086.
2.
de Groot, N . , P a n e t , A. and Lapidot, Y. (1968) Biochem. Biophys. Reg. Commun. 3_j.1, 37-42.
3.
Vogel, Z . , Z a m i r , A. and E l s on, D. ( 1 9 6 8 ) P r o c . Natl. Acad. S c i . LISA. 6_1_1, 701-707.
4.
Lapidot, Y. and de Groot, N. (1972) P r o g . Nucleic. Acid. Reg. Mol. Biol 1_/2, 189-228.
5.
KSssel, H. and R aj B andar y, V . L . ( 1 9 6 8 ) J . Mol. Biol.
6.
J o s t , Y. and Bock, R . M . ( 1 9 6 8 ) J . Biol. Chem.
7.
Menninger, J . R . , D e e r y , S . , D r a p e r , D. and Walker, Ch. (1974) Biochim. Biophys. Acta. 335, 185-195.
260
3_~5, 539-560.
224, 5866-5873.
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8. de Groot, N., Groner, Y. and Lapidot, Y. (1969) Bioehim. Biophys. Acta. 186, 286-296. 9. Ayuso, M.S. and Heredia, C.F. (1968)Eur. J. Biochem. 7, 111-118. i0. Haenni, A.L. and Chapeville, F. (1966) Biochim. Biophys. Acta. 1!4, 135- 148.
11. Lowry, O.H., Rosebrough, N.J., Farr, A . L . and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 12. Lueas-Lenard, J. and Lipmann, F. (1967)Proe. Natl. Aead. Sei. LlSA.57, 1050-1057. 13. MeCroskey, R . P . , Zasloff, M. and Oehoa, S. (1972) P r o c . Natl. Aead. Sei. LISA. 69, 2451- 2455.
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N-ACETYLPHENYLALANYL-tRNA
SPECIFIC
H Y D R O L A S E IN Y E A S T
M . A . B a j o , M . C . M o r a t i l l a and C . F . I n s t i t u t o de E n z i m o l o g £ a del C . S . I . C . ,
Heredia
F a c u l t a d de M e d i c i n a
de la L l n i v e r s i d a d A u t b n o m a , M a d r i d - 3 4 , S p a i n Received
January
14,1975
SUMMARY: An e n z y m e h a s b e e n found and p a r t i a l l y p u r i f i e d f r o m y e a s t which h y d r o l y z e s N - a e e t y l p h e n y l a l a n y l - t R N A and i s d i f f e r e n t f r o m the w e l l characterized N-substituted aminoacyl-tRNA hydrolase. The enzyme splits the e s t e r bond b e t w e e n N - a e e t y l p h e n y l a l a n i n e and the 3 w - h y d r o x y l g r o u p of the t e r m i n a l n u c l e o t i d e in the tRNA. INTRODUCTION:
E n z y m e s h y d r o l y z i n g both p e p t i d y l - t R N A s and N - s u b s t i -
t u t e d a m i n 0 a c y l - t R N A s h a v e b e e n found in the c y t o p l a s m of both p r o k a r y o t i c (1-4) and e u k a r y o t i e (5-7) c e i l s . C o m m o n c h a r a c t e r i s t i c s
to t h e s e e n z y m e s
a r e t h e i r low l e v e l s of s p e c i f i c i t y with r e s p e c t to p a r t i c u l a r a m i n o a c i d s (1) o r p e p t i d y l r e s i d u e s (8) and t h e i r i n a b i l i t y to a t t a c k u n s u b s t i t u t e d a m i n o a c y l t R N A s {1-3). T h e p r o d u c t s of h y d r o l y s i s a r e the i n t a c t tRNA m o l e c u l e and the c o r r e s p o n d i n g N - s u b s t i t u t e d a m i n o a c i d o r p e p t i d y l r e s i d u e . An e n z y m e with t h e s e c h a r a c t e r i s t i c s
(N-substituted aminoacyl-tRNA hydrolase) was de-
t e c t e d (5) and c r y s t a l l i z e d (6) f r o m y e a s t e x t r a c t s .
We now r e p o r t on the
i d e n t i f i c a t i o n of a d i f f e r e n t e n z y m a t i c a c t i v i t y p r e s e n t in y e a s t which h y d r o l y z e s N - a e e t y l p h e n y l a l a n y l - t R N A m u c h m o r e e f f e c t i v e l y than o t h e r N - a c e t y l ated aminoacyl-tRNAs.
T h i s e n z y m e h a s b e e n p a r t i a l l y p u r i f i e d and s e p a -
r a t e d f r o m the p r e v i o u s i d e n t i f i e d h y d r o l a s e . M A T E R I A L S AND M E T H O D S :
Uniformly labeled
f
~
p4cJ
o b t a i n e d f r o m the R a d i o e h e m i e a l C e n t e r ( A m e r s h a m ,
L-amino
acids were
England). Crude yeast
tRNA w a s p u r c h a s e d f r o m B o e h r i n g e r . T h e c h a r g i n g of tRNA with r a d i o a c t i v e a m i n o a c i d s and the i s o l a t i o n of the l 1 4 0 J a m i n o a c y l - t R N A s w a s done
as described
Acetylation of the [14CJaminoacy1-tRNA was performed
u s i n g a c e t i c a n h y d r i d e (10). T h e e x t e n t of a e e t y l a t i o n w a s c h e c k e d by a l k a l i n 1 t r e a t m e n t of the a c e t y l a t e d [14C] a m i n o a c y l - t R N A f o l l o w e d by p a p e r c h r o Copyright © 1975 by Academic Press, Inc. All rights o f reproduction reserved.
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m a t o g r a p h y (9). In all c a s e s m o r e than 95% a c e t y l a t i o n was a c h i e v e d . P r e p a r a t i o n of the y e a s t e x t r a c t s .
The s t r a i n of y e a s t u s e d was the
hybrid S a c c h a r o m y c e s fraqilis x S a c e h a r o m y c e s dobzhanskii.
Cells were
grown as d e s c r i b e d e l s e w h e r e (9) c o l l e c t e d at the m i d d l e of l o g a r i t h m i c p h a s e by c e n t r i f u g a t i o n , w a s h e d t h r e e t i m e s with cold w a t e r and s t o r e d at -20 ° until u s e . One p a r t of y e a s t was g r o u n d in a cold m o r t a r with two p a r t s of s a n d and the p a s t e was s u s p e n d e d in 1-2 v o l u m e s of a cold b u f f e r
con-
taining 50 mM T r i s - H C l pH 7, 10 mM 2 - m e r c a p t o e t h a n o l , and 10 mM m a g n e s i u m a c e t a t e . The h o m o g e n a t e was c e n t r i f u g e d in the cold at 1 0 , 0 0 0 xg f o r 10 rain. and the p r e c i p i t a t e was d i s c a r d e d . The s u p e r n a t a n t was c e n t r i fuged at 105,000 xg in a S p i n e o u l t r a c e n t r i f u g e at 2 - 5 ° f o r one and a half h o u r . The s u p e r n a t a n t a f t e r this c e n t r i f u g a t i o n was u s e d as s o u r c e of e n z y m e and i s r e f e r r e d to as c r u d e y e a s t s o l u b l e f r a c t i o n . T h i s f r a c t i o n can be s t o r e d at -70 °- f o r s e v e r a l d a y s without s i g n i f i c a n t l o s s of a c t i v i t y . A s s a y f o r e n z y m a t i c a c t i v i t y . The a s s a y m i x t u r e (0.1 ml) c o n t a i n e d , u n l e s s o t h e r w i s e s t a t e d , the following c o m p o n e n t s : 50 mM I m i d a z o l - H C l pH 6 . 5 , 2 . 5 mM m a g n e s i u m a c e t a t e , 2 . 5 mM 2 - m e r c a p t o e t h a n o l , about 8 0 p g oftRNAchargedwithS0pmolesofN-acetyl
[14C]aminoaeids (specificactiv -
ity 140 )~C/pmol) and the a m o u n t of e n z y m e i n d i c a t e d in e a c h c a s e . The r e a c t i o n m i x t u r e s w e r e i n c u b a t e d at 30 ° f o r the i n d i c a t e d p e r i o d s of t i m e . At the end of the i n c u b a t i o n the r e a c t i o n was s t o p p e d by addition of 3 ml of 5 p e r cent cold t r i c h l o r o a c e t i c a c i d . The m i x t u r e s w e r e kept at 0 -° f o r 10 rain, and f i l t e r e d t h r o u g h g l a s s f i b e r p a p e r (Whatman G F / C ) ; the f i l t e r s w e r e w a s h e d t h r e e t i m e s with 3 ml e a c h t i m e of 5 p e r cent cold t r i c h l o r o a c e t i c acid and the r a d i o a c t i v i t y r e m a i n i n g in the f i l t e r s was c o u n t e d in a liquid scintillation spectrometer.
C o n t r o l s without added e n z y m e w e r e r u n i n p a r a l l e l
to e s t i m a t e the s p o n t a n e o u s h y d r o l y s i s of the s u b s t r a t e . P r o t e i n s w e r e d e t e r m i n e d by the m e t h o d of L o w r y et al (11). R E S U L T S AND D I S C U S S I O N : The h y d r o l y s i s of d i f f e r e n t N - s u b s t i t u t e d a m i n o a c y l - t R N A s by a c r u d e y e a s t s o l u b l e f r a c t i o n is shown in F i g 1.
The
r a t e s of h y d r o l y s i s of N - a c e t y l v a l y l - t R N A and N - a c e t y l l e u e y l - t R N A a r e s i m i l a r and c l o s e to t h o s e p r e v i o u s l y r e p o r t e d as being due to the y e a s t N - s u b s t i t u t e d a m i n o a e y l - t R N A h y d r o l a s e (6). H o w e v e r N - a c e t y l p h e n y l a l a n y l - t R N A is h y d r o l y z e d at m u c h h i g h e r r a t e s (10 to 50 t i m e s h i g h e r , depending on the
256
Vol. 63, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r51~ !
E
b
e /
40~ (D
o_ v
]3
t¢
3o~ i
',
I0
2
S
?
20 03
a;
I¢
i -Jl
i
I0
5v ~
"o
,I
T
5
I0
15
z
t
,~
I f
?~
g
".; II\kl o ,,4
20
Time (min.) 20
40
60
80
Fraction number
Fig i. H y d r o l y s i s of N - a c e t y l [ 1 4 4 a m i n o a c y l - t R N A s by the y e a s t s o l u b l e f r a c t i o n . R e a c t i o n s w e r e c a r r i e d odt a s d e s c r i b e d in Mfiez~hgds using 10 511 of y e a s t s o l u b l e f r a c t i o n (35. r~ag p r o t e i n / m l ) . N - a c e t y l - [ C] - pJa~nylalanyltRNA ( ® - - e ) ; N - a c e t y l - [ ± ~ C ] v a l y l - t R N A ( o - - o ) ; N - a c e t y l - [ i 4 C ] l e u e y l - t R N A (x--x). Fig 2. F r a c t i o n a t i o n of the y e a s t h y d r o l a s e s on D E A E - S e p h a d e x A-50 c o l u m n . A c o l u m n of D E A E - S e p h a d e x A-50 (2.5 x 14 em) was e q u i l i b r a ted with 50 mM T r i s - H C l , pH 7 c o n t a i n i n g 10 mM 2 - m e r c a p t o e t h a n o l and 10 mM m a g n e s i u m a c e t a t e . The c o l u m n was loaded with 15 ml of c r u d e y e a s t s o l u b l e f r a c t i o n (33 mg p r o t e i n / m l ) , w a s h e d with 65 ml o f the e q u i l i b r a t i o n b u f f e r , and e l u t e d with a l i n e a r g r a d i e n t of 0.1 M - 0 . 4 M T r i s - H C 1 pH 7 c o n t a i n i n g 10 mM 2 - m e r e a p t o e t h a n o l and 10 mM m a g n e s i um a c e t a t e (total b u f f e r 500 ml). The flow r a t e was a d j u s t e d to 0 . 5 ml p e r m i n . and f r a c t i o n s of 6 ml w e r e c o l l e c t e d . A c t i v i t y in the fra~Lonst ~ was d e t e r m i n e d in 10 pl o r 50 pl a l i q u o t z . u s i n g e i t h e r N - a c e t y l - [ CJ p h e n y l a l a n y l - t R N A ( . - - e ) o r N - a c e t y l [14C] v a l y l - tRNA ( o - - o ) r e s p e c t i v e ly a s s u b s t r a t e s .
d i f f e r e n t e x t r a c t s ) . T h i s fact c o n t r a s t s with the a p p a r e n t low s p e c i f i c i t y of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e t o w a r d s p a r t i c u l a r a m i no a c i d s r e s i d u e s (6) and s u g g e s t e d the e x i s t e n c e in y e a s t of a d i f f e r e n t e n z y m a t i c a c t i v i t y acting p r e f e r e n t i a l l y on N - a c e t y l p h e n y l a l a n y l - t R N A .
In a
s e a r c h f o r this a c t i v i t y we u s e d the s a m e y e a s t s t r a i n and f o l l o w e d the f i r s t s t e p s of p u r i f i c a t i o n i d e n t i c a l to t h o s e d e s c r i b e d f o r the c r y s t a l l i z a t i o n of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e , which b a s i c a l l y involve
257
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TABLE
I
S e p a r a t i o n of y e a s t h y d r o l a s e s by f r a c t i o n a t i o n with a m m o n i m s u l f a t e
Volume {ml)
Proteins {mg/ml)
Activity p m o l e s / m g p r o t e i n / 1 0 min N-acetyl-
N-aeetyl-
~4C_]P h e - t R N A ~4CI V a l - t RNA
Crude soluble f r a c t i o n
15
33
150
4.5
D E A E - S e p h a d e x A - 5 0 , then 0-60~0 (NH4) 2 SO 4 p r e c i p i t a t e
5
7.7
14
34
60-80~o (NH4) 2 SO 4 p r e c i p i t a t e
5
4.8
1300
0
A c r u d e s o l u b l e f r a c t i o n was c h r o m a t o g r a p h e d on D E A E - S e p h a d e x A-50 as i n -
dicated in F i g 2. The pooled f r a c t i o n s {120 m l ) , e o n t a i n i n g both a c t i v i t i e s , w e r e f r a c t i o n a t e d by adding solid a m m o n i u m s u l f a t e to r e a c h the p e r c e n t a g e s of s a t u r a t i o n i n d i c a t e d . The p r e c i p i t a t e s w e r e d i s s o l v e d in 5 ml of a b u f f e r c o n taining 10 mM T r i s - H C 1 pH 7, 10 mM p o t a s i u m c h l o r i d e , 10 mM 2 - m e r c a p toethanol and 5 mM m a g n e s i u m a c e t a t e . A c t i v i t y was d e t e r m i n e d as s p e c i f i e d in M e t h o d s .
c h r o m a t o g r a p h y on D E A E - S e p h a d e x and f r a c t i o n a t i o n with a m m o n i u m s u l f a t e (6). The only d i f f e r e n c e was that at e a c h step during the p r o c e s s of p u r i f i c a t i o n , the f r a c t i o n s w e r e a n a l y z e d f o r h y d r o l y t i c a c t i v i t i e s using two s u b s t r a t e s : N - a c e t y l [ 1 4 c l v a l y l - t R N A ( t h e ° n e r ° u t i n e l y u s e d i n t h e p u r[i f i c a1 t i o n of the
N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e ) and N - a c e t y l [ 1 4 C ] p h e n y l a l a n y l ,
tRNA. F i g 2 shows the elution p r o f i l e of the a c t i v i t i e s of a c r u d e y e a s t p r e p a r a t i o n a f t e r c h r o m a t o g r a p h y on D E A E - S e p h a d e x . In line with the r e s u l t s in Fig 1, the total a c t i v i t y on N - a c e t y l p h e n y l a l a n y l - t R N A is m u c h g r e a t e r than that on N - a c e t y l v a l y l - t R N A . M o r e o v e r , the p e a k s of a c t i v i t i e s on t h e s e two s u b s t r a t e s a r e not c o i n c i d e n t . S o m e f r a c t i o n s (40 to 45) which e f f e c t i v e l y h y -
258
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BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
%
//
20 :-'----'--~-~/
×
o
I0
20 Time
~0
k 40
L' 50
(rain.)
Fig 3. H y d r o l y s i s of N - a c e t y l - [ ][14C]phenglglanyl-tRNA with p u r i f i e d h y d r o l a s e and r e c h a r g i n g of the tRNA with [ l ~ C ] p h e n y l a l a n i n e . R e a c t i o n m i x t u r e s containing: 50 mM I m i d a z o l pH 6 . 5 , 1 m i V L 2 - m e r c a p t o e t h a n o l , 1 mM m a g n e s i u m a c e t a t e , 70 p m o l e s of N - a c e t y l -[±'~C] p h e n y l a l a n y l tRNA ( 2 1 , 0 0 0 c p m , s p . a c t . 140 ] a C / ) ~ m o l ) with ( I - - i ) o r without (zX__Zx) h y d r o l a s e p r e p a r a t i o n (20 jal of the 60-80% a m m o n i u m s u l f a t e f r a c t i o n of T a b l e I) w e r e i n c u b a t e d at 30 °-. A f t e r 20 rain i n c u b a t i o n , 50 j~l of a x t u r e c o n t a i n i n g 20 mM A T P , 100 mM m a g n e s i u m a c e t a t e , 0 . 1 5 mM C] p h e n y l a l a n i n e (Sp. a c t i v i t y 116 pO/Mmol) and 20 p l of c r u d e y e a s t s o l u b l e f r a c t i o n ( p r e v i o u s l y p a s s e d t h r o u g h S e p h a d e x G-25) as s o u r c e of p h e n y l a l a n y l - t R N A s y n t h e t a s e w e r e added and the i n c u b a t i o n was c o n t i n u e d . At the t i m e i n d i c a t e d acid i n s o l u b l e c o u n t s w e r e d e t e r m i n e d as i n d i c a t e d in M e t h o d s . C o n t r o l s with on equal a m o u n t ofn~teacylated y e a s t tRNA ( 2 . 6 A,~r_,~ units) ( o - - o ) i n s t e a d of N - a c e t y l [~ "C] p h e n y l a l a n y l /,ou rLm . . .
~
t R N A w e r e run in parallel in reactlons a m o u n t of hydrolase preparation.
mixtures containing
the s a m e
d r o l y z e N - a c e t y l p h e n y l a l a n y l - t R N A have v e r y low o r no d e t e c t a b l e a c t i v i t y on N - a c e t y l v a l y l - t R N A . T h e s e r e s u l t s c o n t r a s t with the a p p a r e n t equal e f f e c t i v i t y of the y e a s t N - s u b s t i t u t e d a m i n o a c y l - t R N A h y d r o l a s e to a t t a c k e i t h e r of t h e s e two s u b s t r a t e s (6). F u r t h e r s e p a r a t i o n of the e n z y m e acting p r e f e r e n t i a l l y on N - a c e t y l p h e n y l a l a n y l - t R N A f r o m the h y d r o l a s e p r e v i o u s l y r e p o r t e d (6) can be a c h i e v e d by f r a c t i o n a t i o n of the p o o l e d f r a c t i o n s f r o m the D E A E - S e p h a d e x c h r o m a t o g r a p h y , c o n t a i n i n g both a c t i v i t i e s , with a m m o n i u m s u l f a t e .
The r e s u l t s in
T a b l e I show that as r e p o r t e d (6) the N - s u b s t i t u t e d a m i n o a e y l - t R N A
259
hydro-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lase p r e c i p i t a t e s with 60% s a t u r a t i o n of ammonium sul fat e. In the 60-80% ammonium sulfate p r e c i p i t a t e t h e r e is the hydrolytic activity v e r y active on N-acetylphenylalanyl-tRNA but which does not hydrol yze N - a c e t y l v a l y l tRNA to any a p p r e c i a b l e extent. P r e l i m i n a r y e x p e r i m e n t s have shown that this f r a c t i o n n e i t h e r h y d r o l y z e s phenylalanyl-tRNA nor the N-aoetylated d e r i v a t i v e s of leucyl_ and methionyl-tRNAs. A study on the speci fi ci t y of this enzyme is now in p r o g r e s s . The r e a c t i o n c a t a l yz e d by this enzym e involves the cleavage of the e s t e r bond between N-acetylphenylalanine and the 31-hydroxyl group of the 3 f t e r m i n a l nucleotide in the tRNA. This has been d e m o n s t r a t e d by r e c h a r g ing the deacylated N-acetylphenylalanyl-tRNA, by the action of the hydrolase, with r_[14Cj phenylalanine (Fig. 3). As it is shown in the F i g u r e the tRNA could be co m pl et el y r e c h a r g e d . The e x c c e s of charging o v e r the input counts is p r e s u m a b l y due to the f r e e tRNA s pe ci fi c for phenylalanine contaminating the p r e p a r a t i o n s of N-acetylphenylalanyl-tRNA. The r ol e of this enzym e in the cell, although still unknown, might be r e l a t e d in someway with the initiation of protein b i o s y n t h e s i s . In this respect it is imp o r tan t to note that N-aeetylphenylalanyl-tRNA acts as analogue of the N - f o r m y l m e t h i o n y l - t R N A F and is r e c o g n i z e d by the p r o k a r y o t i c i n i t i a tim f a c t o r IF 2 or its equivalent in e u k a r y o t i c cells (12 ,J 13). ACKNOWLEDGEMENT S t This work was supported by a grant f r o m the Fondo National pa r a el D e s a r r o l l o de la Investigacibn Cient{fica. REFERENCES 1.
Cuzin, F . , K r e t c h m e r , N . , G r e e n b e r g , R . E . , Hurwitz, R. and Chapeville, F. ( 1 9 6 7 ) P r o c . NatL Acad. S e i . LISA. 5_8, 2079-2086.
2.
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