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The reversal by adenosine triphosphate of ethionine-induced inhibition of protein synthesis
PRELIMINARY NOTES
649
PN 6061
The reversal by adenosine triphosphate of ethionine-induced inhibition of protein synthesis Ethionine, the ethyl analogue of methionine, has been used for several years for the inhibition of protein synthesis in the liver and other organs of tissues (c/. refs. i, 2). Although originally considered to inhibit protein synthesis by virtue of its action as a metabolic antagonist of methionine ~, the exact mechanism of inhibition has remained obscure. Recent studies by SHELL4 have shown that ethionine administration leads to a rapid fall in the concentration of ATP in the liver and that this effect is counteracted b y the injection of adenine or ATP. These results suggested the possibility that ethionine m a y inhibit protein synthesis indirectly through an effect upon the hepatic ATP level rather than directly, an hypothesis first suggested by STEKOL and coworkers in 19555. These considerations led to the present study of the effect of ATP administration upon the incorporation in vitro of radioactive leucine into protein in a ribosomal system prepared from the livers of animals also injected with ethionine. Experimental white Wistar (Carworth Farms) female rats weighing from 18o220 g were fasted overnight and were then injected with DL-ethionine with or without ATP, both in aqueous solution. Each rat received I m g of the appropriate compound per g body wt. in two divided doses at zero and I h, the ethionine being administered intraperitoneally and the ATP subcutaneously. Control animals received equal volumes of water in place of ethionine or ATP. The animals were sacrificed at 5 h by a blow on the head and ribosomes were prepared from their livers by the deoxycholate method of KORNER~ as modified by WETTSTEIN et al. 7. The livers were quickly homogenized in 2.3 vol. of ice-cold medium A of LITTLEFIELD AND KELLERs and centrifuged at 15 ooo rev./min for 15 min to remove mitochondria, nuclei, whole cells and debris. To 5 ml of the supernatant were added 0. 5 ml of IO % deoxycholate and 5 ml of medium A and the resulting suspension was centrifuged at 40 ooo rev./ min for 12o rain. The ribosome pellet was rinsed twice with 2 ml each of medium A and resuspended in a 1.5 ml of the same solution. This suspension could be stored at - - 4 °0 for at least I week without loss of incorporating activity. It was usually used within 24 h after its preparation. The pH-5 fraction was prepared fresh just before use by the method of KELLER AND ZAMECNIK9 without the use of medium B and with I instead of 3 vol. of medium A. All centrifugations were performed in a Spinco model L ultracentrifuge with No.-4o rotor. As seen in Table I, ribosomes prepared from the liver of female rats injected 5 h previously with BL-ethionine had only about IO % of the incorporating activity of those from control animals. The administration of ATP to ethionine-treated rats counteracted completely the marked inhibitory effect of ethionine upon leucine incorporation into protein. A T P injected into control rats caused only a slight increase of the extent of incorporation. These findings are very constant and have been obtained in several experiments as summarized in Table II. A similar reversing effect of ATP administration has been found in the case of the inhibitory action of ethionine on the increase in 2-acetylaminofluorene hydroxylase activity in the liver in young rats following the injection of methylcholanthrene 1°. Previous results showed that the main site of the block in leucine incorporation Biochim. Biophys. Acta, 61 (1962) 6 4 9 - 6 5 I
650
PRELIMINARY NOTES TABLE I
INCORPORATION OF RADIOACTIVE L-LEUCINE INTO THE PROTEIN OF LIVER RIBOSOMAL SYSTEM FROM ETHIONINE, ETHIONINE- AND ATP-TREATED OR CONTROL FEMALE RATS T h e i n c u b a t i o n m i x t u r e c o n t a i n e d , in a t o t a l vol. of i ml, r i b o s o m e s (i.5 m g of p r o t e i n ) , p H 5- fract i o n (2. 5 m g of p r o t e i n ) , o.ooi 5 M ATP, o.ooo 5 M GTP, o.oI M p h o s p h o e n o l p y r u v a t e , 3o/zg p y r u v a t e k i n a s e (EC 2.7.I.4O), o.oo12 M KC1, o.o25 M T r i s - H C 1 buffe r (pH 7.6), o.oo 5 M MgC12 a n d o.2/zC of L-[14C]leucine u n i f o r m l y l a b e l e d w i t h a specific a c t i v i t y of 7.14 m C / m m o l e ( N u c l e a r Chicago). The s a m p l e s were i n c u b a t e d for 60 rain a t 37.5 °. The r e a c t i o n w a s s t o p p e d w i t h 2 m l of i o % t r i c h l o r a c e t i c acid c o n t a i n i n g i o m g of n o n - r a d i o a c t i v e l e uc i ne p e r ml. The s a m p l e s w e r e w a s h e d t w i c e w i t h cold lO % t r i c h l o r o a c e t i c a c i d , e x t r a c t e d once w i t h e t h a n o l - e t h e r (3 : I) for 5 rain a t 6o ° a n d once w i t h i o % t r i c h l o r o a c e t i c a c i d for 15 m i n a t 9 °° a n d w a s h e d once each w i t h e t h a n o l , e t h a n o l - e t h e r a n d ether. T h e y w e re p l a t e d a n d c o u n t e d i n a t h i n - w i n d o w gas-flow c o u n t e r w i t h an efficienc y of 25 % for 14C.
Tissue components*
Radioactivity in protein counts[rain/rag
Dil]erence /rum control %
RC+SC RE+SC REA+SEA RC+SA RC + S E A RE+SEA REA+SE R E A + SA Zero-time control
593~z36"* 54:t: I 577=[=71 699=E94 7 8 2 i 27 63i3 682±42 604 4- 76 4
---91 --3 + 18 + 32 --89 +15 + 2 ioo
* RC, R i b o s o m e s f r o m c o n t r o l r a t ; R E , R i b o s o m e s f r o m e t h i o n i n e - t r e a t e d a n i m a l ; R E A , R i b o s o m e s f r o m e t h i o n i n e - A T P - t r e a t e d a n i m a l ; SC, p H - 5 f r a c t i o n from c o n t r o l a n i m a l ; S EA , p H - 5 f r a c t i o n f r o m e t h i o n i n e - A T P - t r e a t e d a n i m a l ; SE, p H - 5 f r a c t i o n f r o m e t h i o n i n e - t r e a t e d a n i m a l ; SA, p H - 5 f r a c t i o n f r o m A T P - t r e a t e d a n i m a l . ** E a c h v a l u e r e p r e s e n t s t h e a v e r a g e of 4 i n c u b a t e d s a m p l e s f r o m 2 a n i m a l s 4- t h e s t a n d a r d e r r o r of t h e m e a n . TABLE II EFFECT OF ETHIONINE AND A T P ADMINISTRATION TO FEMALE RATS UPON THE INCORPORATION OF RADIOACTIVE LEUCINE INTO A LIVER RIBOSOMAL-SUPERNATANT SYSTEM I n c u b a t i o n c o n d i t i o n s were t h e s a m e as in T a b l e I. I n e a c h e x p e r i m e n t , one z e r o - t i m e s a m p l e was run and it never had more than 5 counts/rain. Number o] animals* Control Ethionine Ethionine + ATP Control + ATP
2o 21 12 6
Relative speei/ic activity o] protein*" lOO 21 :f: 3 *"* lO3 4-9 i 15 :t: 3
* D u p l i c a t e f l a s k s were r u n on each a n i m a l . ** I n e a c h e x p e r i m e n t t h e specific a c t i v i t y of t h e c o n t r o l is c o n s i d e r e d as i o o a n d t h e o t h e r g r o u p s are e x p r e s s e d r e l a t i v e t o t h e c o n t r o l v a l u e s . The o b s e r v e d r a d i o a c t i v i t y i n t h e c o n t r o l s r a n g e d f r o m 6OO-lOOO c o u n t s / r a i n . *** M e a n -4- s t a n d a r d error of t h e m e a n .
into protein in preparations from ethionine-treated rats was in the microsomes 1. The results in Table I indicate that in the ribosomal system used in the present study, the inhibition is localized to the ribosomes and not to the pH-5 fraction. ATP appears to have its major site of reversal at the same locus. B i o c h i m . B i o p h y s . A c t a , 61 (1962) 649-651
PRELIMINARY NOTES
651
The administration of adenine in place of ATP is equally effective in counteracting the inhibitory effect of ethionine on the incorporating system. However, ATP has been used much more frequently than adenine in the study since the nucleotide is much less toxic than the free purine. These findings concerning the efficacy of adenine or ATP in counteracting an effect of ethionine, coupled with the observed drop in hepatic ATP level4, lend considerable support to the hypothesis that some of the acute effects of ethionine in animals are secondary to the trapping of adenine by ethionineS, n. This is considered to be due to the relatively rapid formation of S-adenosylethionine from ethionine and ATP via the methionine-activating enzyme coupled with the poor utilization of the activated ethionine in other metabolic reactions. It is tentatively concluded from these results that ethionine, as suspected s, does not inhibit hepatic protein synthesis directly but only indirectly via its effect upon the ATP concentration in the liver. This research was supported by research grants from the Life Insurance Medical Research Fund, United States Public Health Service (C 6074 and A 559o), the Beaver County Cancer Society and a United States Public Health Service Training Grant (2G-I35).
Department o/ Pathology, University o~ Pittsburgh School o/ Medicine, Pittsburgh Pa. (U.S.A.)
SAUL VILLA-TREVINO* EMMANUEL ]VARBER
1 E. FARBER AND S. M. CORBAN, J. Biol. Chem., 233 (1958) 625. E. FARBER, Advances in Cancer Research, 7 (I962), in the press. 3 M. V. SIMPSON, E. FARBER AND I-I. TARVER, J. Biol. Chem., 182 (195 o) 81. 4 K. H. SHULL, J. Biol. Chem., 237 (1962) PCI734. 5 j . A. STEKOL, Abstract o/ papers o/ the I29th meeting o/ the Am. Chem. Sot., Am. Chem. Soc., Washington, D.C., 1955, p. 4 C. 6 A. KORNER, Biochim. Biophys. Acta, 35 (1959) 554. 7 F. \~ETTSTEIN, T. STAEHELIN AND H. NOLL, personal c o m m u n i c a t i o n . s \V. J. LITTLEFIELO ANn E. B. KELLER, J. Biol. Chem., 224 (1957) 13. 8 j~. B. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 221 (1956) 45. 10 j. "~V. CRAMER, J. A. MILLER AND E. C. MILLER, J. Biol. Chem., 235 (196o) 250. 11 G. SCHMIDT, K. SERAIDARIAN, L. M. GREENBAUM, M. D. H1CKEY AND S. J. THANNHAUSER, Bioehim. Biophys. Acta, 20 (1956) 135.
Received July I3th, 1962 * Rockefeller F o u n d a t i o n Fellow, 1958 to 1962, and Sarah Mellon Scaife Fellow, 1962.
Biochim. Biophys. Acta, 61 (1962) 649-65I
649
PN 6061
The reversal by adenosine triphosphate of ethionine-induced inhibition of protein synthesis Ethionine, the ethyl analogue of methionine, has been used for several years for the inhibition of protein synthesis in the liver and other organs of tissues (c/. refs. i, 2). Although originally considered to inhibit protein synthesis by virtue of its action as a metabolic antagonist of methionine ~, the exact mechanism of inhibition has remained obscure. Recent studies by SHELL4 have shown that ethionine administration leads to a rapid fall in the concentration of ATP in the liver and that this effect is counteracted b y the injection of adenine or ATP. These results suggested the possibility that ethionine m a y inhibit protein synthesis indirectly through an effect upon the hepatic ATP level rather than directly, an hypothesis first suggested by STEKOL and coworkers in 19555. These considerations led to the present study of the effect of ATP administration upon the incorporation in vitro of radioactive leucine into protein in a ribosomal system prepared from the livers of animals also injected with ethionine. Experimental white Wistar (Carworth Farms) female rats weighing from 18o220 g were fasted overnight and were then injected with DL-ethionine with or without ATP, both in aqueous solution. Each rat received I m g of the appropriate compound per g body wt. in two divided doses at zero and I h, the ethionine being administered intraperitoneally and the ATP subcutaneously. Control animals received equal volumes of water in place of ethionine or ATP. The animals were sacrificed at 5 h by a blow on the head and ribosomes were prepared from their livers by the deoxycholate method of KORNER~ as modified by WETTSTEIN et al. 7. The livers were quickly homogenized in 2.3 vol. of ice-cold medium A of LITTLEFIELD AND KELLERs and centrifuged at 15 ooo rev./min for 15 min to remove mitochondria, nuclei, whole cells and debris. To 5 ml of the supernatant were added 0. 5 ml of IO % deoxycholate and 5 ml of medium A and the resulting suspension was centrifuged at 40 ooo rev./ min for 12o rain. The ribosome pellet was rinsed twice with 2 ml each of medium A and resuspended in a 1.5 ml of the same solution. This suspension could be stored at - - 4 °0 for at least I week without loss of incorporating activity. It was usually used within 24 h after its preparation. The pH-5 fraction was prepared fresh just before use by the method of KELLER AND ZAMECNIK9 without the use of medium B and with I instead of 3 vol. of medium A. All centrifugations were performed in a Spinco model L ultracentrifuge with No.-4o rotor. As seen in Table I, ribosomes prepared from the liver of female rats injected 5 h previously with BL-ethionine had only about IO % of the incorporating activity of those from control animals. The administration of ATP to ethionine-treated rats counteracted completely the marked inhibitory effect of ethionine upon leucine incorporation into protein. A T P injected into control rats caused only a slight increase of the extent of incorporation. These findings are very constant and have been obtained in several experiments as summarized in Table II. A similar reversing effect of ATP administration has been found in the case of the inhibitory action of ethionine on the increase in 2-acetylaminofluorene hydroxylase activity in the liver in young rats following the injection of methylcholanthrene 1°. Previous results showed that the main site of the block in leucine incorporation Biochim. Biophys. Acta, 61 (1962) 6 4 9 - 6 5 I
650
PRELIMINARY NOTES TABLE I
INCORPORATION OF RADIOACTIVE L-LEUCINE INTO THE PROTEIN OF LIVER RIBOSOMAL SYSTEM FROM ETHIONINE, ETHIONINE- AND ATP-TREATED OR CONTROL FEMALE RATS T h e i n c u b a t i o n m i x t u r e c o n t a i n e d , in a t o t a l vol. of i ml, r i b o s o m e s (i.5 m g of p r o t e i n ) , p H 5- fract i o n (2. 5 m g of p r o t e i n ) , o.ooi 5 M ATP, o.ooo 5 M GTP, o.oI M p h o s p h o e n o l p y r u v a t e , 3o/zg p y r u v a t e k i n a s e (EC 2.7.I.4O), o.oo12 M KC1, o.o25 M T r i s - H C 1 buffe r (pH 7.6), o.oo 5 M MgC12 a n d o.2/zC of L-[14C]leucine u n i f o r m l y l a b e l e d w i t h a specific a c t i v i t y of 7.14 m C / m m o l e ( N u c l e a r Chicago). The s a m p l e s were i n c u b a t e d for 60 rain a t 37.5 °. The r e a c t i o n w a s s t o p p e d w i t h 2 m l of i o % t r i c h l o r a c e t i c acid c o n t a i n i n g i o m g of n o n - r a d i o a c t i v e l e uc i ne p e r ml. The s a m p l e s w e r e w a s h e d t w i c e w i t h cold lO % t r i c h l o r o a c e t i c a c i d , e x t r a c t e d once w i t h e t h a n o l - e t h e r (3 : I) for 5 rain a t 6o ° a n d once w i t h i o % t r i c h l o r o a c e t i c a c i d for 15 m i n a t 9 °° a n d w a s h e d once each w i t h e t h a n o l , e t h a n o l - e t h e r a n d ether. T h e y w e re p l a t e d a n d c o u n t e d i n a t h i n - w i n d o w gas-flow c o u n t e r w i t h an efficienc y of 25 % for 14C.
Tissue components*
Radioactivity in protein counts[rain/rag
Dil]erence /rum control %
RC+SC RE+SC REA+SEA RC+SA RC + S E A RE+SEA REA+SE R E A + SA Zero-time control
593~z36"* 54:t: I 577=[=71 699=E94 7 8 2 i 27 63i3 682±42 604 4- 76 4
---91 --3 + 18 + 32 --89 +15 + 2 ioo
* RC, R i b o s o m e s f r o m c o n t r o l r a t ; R E , R i b o s o m e s f r o m e t h i o n i n e - t r e a t e d a n i m a l ; R E A , R i b o s o m e s f r o m e t h i o n i n e - A T P - t r e a t e d a n i m a l ; SC, p H - 5 f r a c t i o n from c o n t r o l a n i m a l ; S EA , p H - 5 f r a c t i o n f r o m e t h i o n i n e - A T P - t r e a t e d a n i m a l ; SE, p H - 5 f r a c t i o n f r o m e t h i o n i n e - t r e a t e d a n i m a l ; SA, p H - 5 f r a c t i o n f r o m A T P - t r e a t e d a n i m a l . ** E a c h v a l u e r e p r e s e n t s t h e a v e r a g e of 4 i n c u b a t e d s a m p l e s f r o m 2 a n i m a l s 4- t h e s t a n d a r d e r r o r of t h e m e a n . TABLE II EFFECT OF ETHIONINE AND A T P ADMINISTRATION TO FEMALE RATS UPON THE INCORPORATION OF RADIOACTIVE LEUCINE INTO A LIVER RIBOSOMAL-SUPERNATANT SYSTEM I n c u b a t i o n c o n d i t i o n s were t h e s a m e as in T a b l e I. I n e a c h e x p e r i m e n t , one z e r o - t i m e s a m p l e was run and it never had more than 5 counts/rain. Number o] animals* Control Ethionine Ethionine + ATP Control + ATP
2o 21 12 6
Relative speei/ic activity o] protein*" lOO 21 :f: 3 *"* lO3 4-9 i 15 :t: 3
* D u p l i c a t e f l a s k s were r u n on each a n i m a l . ** I n e a c h e x p e r i m e n t t h e specific a c t i v i t y of t h e c o n t r o l is c o n s i d e r e d as i o o a n d t h e o t h e r g r o u p s are e x p r e s s e d r e l a t i v e t o t h e c o n t r o l v a l u e s . The o b s e r v e d r a d i o a c t i v i t y i n t h e c o n t r o l s r a n g e d f r o m 6OO-lOOO c o u n t s / r a i n . *** M e a n -4- s t a n d a r d error of t h e m e a n .
into protein in preparations from ethionine-treated rats was in the microsomes 1. The results in Table I indicate that in the ribosomal system used in the present study, the inhibition is localized to the ribosomes and not to the pH-5 fraction. ATP appears to have its major site of reversal at the same locus. B i o c h i m . B i o p h y s . A c t a , 61 (1962) 649-651
PRELIMINARY NOTES
651
The administration of adenine in place of ATP is equally effective in counteracting the inhibitory effect of ethionine on the incorporating system. However, ATP has been used much more frequently than adenine in the study since the nucleotide is much less toxic than the free purine. These findings concerning the efficacy of adenine or ATP in counteracting an effect of ethionine, coupled with the observed drop in hepatic ATP level4, lend considerable support to the hypothesis that some of the acute effects of ethionine in animals are secondary to the trapping of adenine by ethionineS, n. This is considered to be due to the relatively rapid formation of S-adenosylethionine from ethionine and ATP via the methionine-activating enzyme coupled with the poor utilization of the activated ethionine in other metabolic reactions. It is tentatively concluded from these results that ethionine, as suspected s, does not inhibit hepatic protein synthesis directly but only indirectly via its effect upon the ATP concentration in the liver. This research was supported by research grants from the Life Insurance Medical Research Fund, United States Public Health Service (C 6074 and A 559o), the Beaver County Cancer Society and a United States Public Health Service Training Grant (2G-I35).
Department o/ Pathology, University o~ Pittsburgh School o/ Medicine, Pittsburgh Pa. (U.S.A.)
SAUL VILLA-TREVINO* EMMANUEL ]VARBER
1 E. FARBER AND S. M. CORBAN, J. Biol. Chem., 233 (1958) 625. E. FARBER, Advances in Cancer Research, 7 (I962), in the press. 3 M. V. SIMPSON, E. FARBER AND I-I. TARVER, J. Biol. Chem., 182 (195 o) 81. 4 K. H. SHULL, J. Biol. Chem., 237 (1962) PCI734. 5 j . A. STEKOL, Abstract o/ papers o/ the I29th meeting o/ the Am. Chem. Sot., Am. Chem. Soc., Washington, D.C., 1955, p. 4 C. 6 A. KORNER, Biochim. Biophys. Acta, 35 (1959) 554. 7 F. \~ETTSTEIN, T. STAEHELIN AND H. NOLL, personal c o m m u n i c a t i o n . s \V. J. LITTLEFIELO ANn E. B. KELLER, J. Biol. Chem., 224 (1957) 13. 8 j~. B. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 221 (1956) 45. 10 j. "~V. CRAMER, J. A. MILLER AND E. C. MILLER, J. Biol. Chem., 235 (196o) 250. 11 G. SCHMIDT, K. SERAIDARIAN, L. M. GREENBAUM, M. D. H1CKEY AND S. J. THANNHAUSER, Bioehim. Biophys. Acta, 20 (1956) 135.
Received July I3th, 1962 * Rockefeller F o u n d a t i o n Fellow, 1958 to 1962, and Sarah Mellon Scaife Fellow, 1962.
Biochim. Biophys. Acta, 61 (1962) 649-65I