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Specific incorporation of adenosine-5′-phosphate-32P into ribonucleic acid in rat liver homogenates
VOL. 20 (1956)
PRELIMINARY NOTES
445
Although peptide I (Table I) and its di-iodo derivative were the most sensitive towards fraction PEP/B16, the same weight of certain cruder preparations of the peptidase (on the basis of their higher contents of protease contaminant), while releasing less tyrosine from peptide I, readily hydrolysed peptide III (Cbz.-Gly.-L-Phe.) under comparable conditions. It is, therefore, considered that there are at least two thyroid peptidases of the carboxypeptidase type, both active at p H 3.5. In addition to peptide VII the protease fraction hydrolysed the tripeptide, L-CySH.-L-Tyr.L-isoLeu., at the cysteinyl link and, to a lesser extent, L-Tyr.-L-CySH. Fission of peptide VII could also be demonstrated at p H 7.4 (phosphate buffer) to about the same degree as at pH 3.5, if oxygen was excluded during incubation. Crystalline pepsin has been reported to hydrolyse peptides of cysteine and tyrosin~. However, such peptidase action in the thyroid may not be inherent in the protease molecule. Thus, although o.2 mg of each of four fractions obtained by electrophoresis of a sample of protease (55 o units/mg), by the procedure reported previously~, hydrolysed cysteinyl-tyrosine to approximately the same extent, the fractions assayed at 16oo, 93 o, 700 and 13o protease units/mg. The enzyme fractions PRO/B16 and PEP/B16 were also tested on rat thyroglobulin labelled in vivo with 1eli. Pooled thyroids from 5-7 rats, each of which had been injected 24-42 hours previously with 3o/~c carrier-free 1SlI, were blended with saline, and the supernatant brought to 45 % saturation with ammonium sulphate. The solution of the precipitate, after dialysis, was usually heated for 4 minutes at 80 ° C to reduce proteolytic activity and precipitated with acetone at - - 5 ° C (to remove traces of free iodo-amino acids). Aqueous solutions of this precipitate and the enzyme fractions were incubated at 37°C and pH 3.5 for 2 or I6 hours. Chromatograms (n-pentanol-propionic acid-water (2o:3:i5)) and radioautograms were then prepared. The main qualitative difference between the two enzyme fractions so far observed in such experiments is t h a t the peptidase releases more radioactivity in the form of di-iodotyrosine than of mono-iodotyrosine, whereas the reverse holds for the protease. We are indebted to the Australian National Health and Medical Research Council for a graut towards the cost of the investigation; also to Dr. J. M. SWAN of the Commonwealth Scientific and Industrial Research Organization, Melbourne, and, through him, to Dr, J. WATSON of the Courtauld Research Laboratory, England, for certain of the tyrosine-containing peptides.
Department o/Biochemistry, University o/Melbourne (Australia)
W. G. LAVER V. l~I. TRIKOJUS
x W. G. LAVER AND V. M. TRIKOJUS, Biochim. Biophys. Acta, 16 (1955) 592. I M. T. McQUZLLAN, P. G. STANLEY ArCDV. M. TRIKOJUS, Australian J. Biol. Sci., 7 (1954) 319 • s M. L. ANSON, J. Gen. Physiol., 22 (1938) 79. 4 C. S. HANE$, F. J. R. HIRD AND F. A. ISHERWOOD, Nature, 166 (195o) 288. 6 C. R. HARINGTON AND R. V. PITT RIVERS, Biochem. J., 38 (1944) 417 . Received February Ifth, 1956
Specific incorporation of adenosine-5'-phosphate-~P into ribonucleic acid in rat liver homogenate$* We wish to report the demonstration t h a t a nucleoside-5'-phosphate can be incorporated specifically into ribonucleic acid (RNA) without randomization or exchange of its phosphorus. Previous experiments with whole animals 1 and with tissue slices and cell suspensions2, had shown that when either 3' or 5' nucleoside mon0phosphates, labeled with sip, are employed as precursors, the sip is separated from the nucleoside and appears randomly distributed among all four nucleotides obtained from the labeled RNA. These results are explained by the impermeability of animal cells to mononucleotides, which are presumably cleaved at the cell membranes~. The development of cell-free systems that are capable of incorporating labeled precursors into RNA 8 has provided the tool for testing whether an intact nucleoside-5'-phosphate could be incorporated as such into RNA. When adenosine-5'-82phosphate (AM32P, obtained from the liver acid-soluble fraction be partially hepatectomized rats treated with inorganic sap) was incubated with the "cytoplasmic fraction" obtained by centrifuging the nuclei from a o.25 M sucrose homogenate of rat liver under conditions in which oxidative phosphorylation was maintained, radioactivity was incorporated * This work was supported in part by a research grant, C-24o9, from the National Cancer Institute, National Institutes of Health, Public Health Service, and in part by ~ grant from the Wisconsin Section of the American Cancer Society.
446
PRELIMINARY NOTES
VOL, 2 0 (1956)
into t h e R N A . Following isolation, t h e R N A was h y d r o l y z e d w i t h diesterase (prepared according to HURST AND BUTLERs f r o m dialyzed* s n a k e v e n o m ) , w h i c h cleaves t h e internucleotide p h o s p h o diester so as to give t h e 5 ' - m o n o n u c l e o t i d e s 5, t h e r a d i o a c t i v i t y w a s c o n c e n t r a t e d a l m o s t entirely in t h e 5 ' - A M P o b t a i n e d f r o m t h e h y d r o l y s a t e (Table I). T h u s t h e i n c u b a t e d nucleotide was incorporated into R N A w i t h o u t loss or r a n d o m i z a t i o n of its p h o s p h o r u s , a n d s h o w s clearly t h a t 5'-nucleotides are t h e a c t u a l i n t e r m e d i a t e s in R N A biosynthesis. R a t liver h o m o g e n i z e d in 0.25 M sucrose, a n d t h e s u p e r n a t a n t from 6oo × g c e n t r i f u g a t i o n was used. A RECOVERED RADIOACTIVITY FROM R N A o. 5 g e q u i v a l e n t of tissue was i n c u b a t e d in air at 3 °° C for 4 ° m i n u t e s in a t o t a l Diestemse hydrolysis Alkalinehydrolysis v o l u m e of 5.o ml including t h e follow(S') (~" + 39 ing c o n c e n t r a t i o n s of: O . o l M glutamate, o.oI M p y r u v a t e , o.004 M f u m a Adenylic acid 92 5-4 rate, o.oi M p h o s p h a t e buffer (pH 7.2), Cytidylic acid 0.6 87. 4 O . o l M m a g n e s i u m chloride, o.o331 Uridylic acid 0. 5 1.2 fructose, o.25.~I sucrose a n d ~ microG u a n y l i c acid o. 4 1.2 mole of AMz2p (specific activity, Inorganic phosphorus 6. I 4.8 6o8,ooo c.p.m./pM) 4. Four flasks were used a n d pooled. Acid-soluble fractions s a n d R N A 7were isolated, e n z y m i c a n d alkaline h y d r o l y s e s were carried o u t 2, a n d t h e nucleotides isolated b y g r a d i e n t elution chrom a t o g r a p h y on D o w e x - i 7. T h e uridylic acid was s e p a r a t e d from c o n t a m i n a t i n g inorganic p h o s p h a t e b y a d s o r p t i o n on charcoal, w a s h i n g w i t h water, a n d elution w i t h lO % pyridine. TABLE I
W h e n t h e s a m e s a m p l e of R N A was h y d r o l y z e d with alkali, which cleaves t h e internucleotide p h o s p h o diester to give a m i x t u r e of t h e m o n o n u c l e o t i d e - 2 ' a n d 3 ' - p h o s p h a t e s , t h e r a d i o a c t i v i t y was localized a l m o s t entirely in t h e cytidylic acid, a n d v e r y little w a s f o u n d in t h e o t h e r t h r e e nucleotides (Table I). T h e s e results s u g g e s t t h a t in t h e p a r t i c u l a r R N A t h a t was labeled u n d e r t h e s e conditions, ,adenosine is a d j a c e n t to cytidine, w i t h t h e radioactive p h o s p h a t e a t t a c h e d to t h e 5 ' - c a r b o n of t h e a d e n o s i n e a n d t h e 3 ' - c a r b o n of t h e cytidine. GOLDWASSER8 h a s v e r y r e c e n t l y reported t h e specific incorporation of 1*C-adenine-labeled A M P into t h e adenylic acid, o b t a i n e d b y alkaline h y d r o l y s i s of R N A , in a pigeon liver h o m o g e n a t e . However, in a similar e x p e r i m e n t u s i n g s ' P - l a b e l e d AMP, he f o u n d t h e label to be d i s t r i b u t e d a m o n g all four nucleotides r e s u l t i n g f r o m alkaline hydrolysis, w i t h t h e r a d i o a c t i v i t y in adenylic acid below t h e a v e r a g e a n d in uridylic acid, a b o v e t h e average. O u r results are also c o m p l e m e n t a r y to t h e e l e g a n t w o r k of GRUNBERG-MANAGO, ORTIZ, AND OCHOA9, w h o h a v e o b t a i n e d RNA-like p o l y n u c l e o t i d e s b y i n c u b a t i o n of n u c l e o s i d e - 5 ' - d i p h o s p h a t e s w i t h a polynucleotide p h o s p h o r y l a s e o b t a i n e d from Azotobacter vinelandii. I n our case, A D P m a y be t h e actual i n t e r m e d i a t e , w i t h t h e 32p of t h e A M P a c t u a l l y i n c o r p o r a t e d into t h e R N A since t h o s y s t e m p r o d u c e d A D P a n d A T P a n d t h e original inorganic p h o s p h o r u s w a s unlabeled. Similar e x p e r i m e n t s w i t h o t h e r nucleotides are b e i n g c o n t i n u e d a n d a m o r e detailed report will be p u b l i s h e d elsewhere.
~/IcArdle Memorial Laboratory, The Medical School, University o[ Wisconsin, Madison, Wis. (U.S.A.)
CHARLES HEIDELBERGER EBERHARD HARBERS** KENNETH C. LEIBMAN Y . TAKAGI
VAN R. POTTER I p. M. ROLL, H. WEINFELD AND G. B. BROWN, Biochim. Biophys. Acta, 13 (I954) 141. 2 K. C. LEIBMAN AND C. HEIDELBERGER, J. Biol. Chem., 216 (1955) 823. 3 V. R. POTTER et al., Biochim. Biophys. Acta, 2o (1956) 439. 4 R. O. HURST AND G. C. BUTLER, J. Biol. Chem., 193 (1951) 91. s W. E. COHN AND E. VOLKIN, J. Biol. Chem., 2o 3 (1953) 319. R. 13. HURLBERT, H. SCHMITZ, A. F. 13RUMM AND V. R. POTTER, J. Biol. Chem., 2o9 (1954) 23. R. 13. HURLBEET AND V. R. POTTER,J. Biol. Chem., I95 (I955) 257. s E. GOLDWASSE.R, J. Am. Chem. Soc., 77 (1955) 6o83. 9 M. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Science, 122 (1955) 9o7. Received March ist, 1956 * W e are g r e a t l y i n d e b t e d to Prof. EUGENE KENNEDY for t h e s u g g e s t i o n of dialyzing t h e crude v e n o m . ** F u l b r i g h t R e s e a r c h Fellow f r o m t h e U n i v e r s i t y of G 6 t t i n g e n , G e r m a n y .
PRELIMINARY NOTES
445
Although peptide I (Table I) and its di-iodo derivative were the most sensitive towards fraction PEP/B16, the same weight of certain cruder preparations of the peptidase (on the basis of their higher contents of protease contaminant), while releasing less tyrosine from peptide I, readily hydrolysed peptide III (Cbz.-Gly.-L-Phe.) under comparable conditions. It is, therefore, considered that there are at least two thyroid peptidases of the carboxypeptidase type, both active at p H 3.5. In addition to peptide VII the protease fraction hydrolysed the tripeptide, L-CySH.-L-Tyr.L-isoLeu., at the cysteinyl link and, to a lesser extent, L-Tyr.-L-CySH. Fission of peptide VII could also be demonstrated at p H 7.4 (phosphate buffer) to about the same degree as at pH 3.5, if oxygen was excluded during incubation. Crystalline pepsin has been reported to hydrolyse peptides of cysteine and tyrosin~. However, such peptidase action in the thyroid may not be inherent in the protease molecule. Thus, although o.2 mg of each of four fractions obtained by electrophoresis of a sample of protease (55 o units/mg), by the procedure reported previously~, hydrolysed cysteinyl-tyrosine to approximately the same extent, the fractions assayed at 16oo, 93 o, 700 and 13o protease units/mg. The enzyme fractions PRO/B16 and PEP/B16 were also tested on rat thyroglobulin labelled in vivo with 1eli. Pooled thyroids from 5-7 rats, each of which had been injected 24-42 hours previously with 3o/~c carrier-free 1SlI, were blended with saline, and the supernatant brought to 45 % saturation with ammonium sulphate. The solution of the precipitate, after dialysis, was usually heated for 4 minutes at 80 ° C to reduce proteolytic activity and precipitated with acetone at - - 5 ° C (to remove traces of free iodo-amino acids). Aqueous solutions of this precipitate and the enzyme fractions were incubated at 37°C and pH 3.5 for 2 or I6 hours. Chromatograms (n-pentanol-propionic acid-water (2o:3:i5)) and radioautograms were then prepared. The main qualitative difference between the two enzyme fractions so far observed in such experiments is t h a t the peptidase releases more radioactivity in the form of di-iodotyrosine than of mono-iodotyrosine, whereas the reverse holds for the protease. We are indebted to the Australian National Health and Medical Research Council for a graut towards the cost of the investigation; also to Dr. J. M. SWAN of the Commonwealth Scientific and Industrial Research Organization, Melbourne, and, through him, to Dr, J. WATSON of the Courtauld Research Laboratory, England, for certain of the tyrosine-containing peptides.
Department o/Biochemistry, University o/Melbourne (Australia)
W. G. LAVER V. l~I. TRIKOJUS
x W. G. LAVER AND V. M. TRIKOJUS, Biochim. Biophys. Acta, 16 (1955) 592. I M. T. McQUZLLAN, P. G. STANLEY ArCDV. M. TRIKOJUS, Australian J. Biol. Sci., 7 (1954) 319 • s M. L. ANSON, J. Gen. Physiol., 22 (1938) 79. 4 C. S. HANE$, F. J. R. HIRD AND F. A. ISHERWOOD, Nature, 166 (195o) 288. 6 C. R. HARINGTON AND R. V. PITT RIVERS, Biochem. J., 38 (1944) 417 . Received February Ifth, 1956
Specific incorporation of adenosine-5'-phosphate-~P into ribonucleic acid in rat liver homogenate$* We wish to report the demonstration t h a t a nucleoside-5'-phosphate can be incorporated specifically into ribonucleic acid (RNA) without randomization or exchange of its phosphorus. Previous experiments with whole animals 1 and with tissue slices and cell suspensions2, had shown that when either 3' or 5' nucleoside mon0phosphates, labeled with sip, are employed as precursors, the sip is separated from the nucleoside and appears randomly distributed among all four nucleotides obtained from the labeled RNA. These results are explained by the impermeability of animal cells to mononucleotides, which are presumably cleaved at the cell membranes~. The development of cell-free systems that are capable of incorporating labeled precursors into RNA 8 has provided the tool for testing whether an intact nucleoside-5'-phosphate could be incorporated as such into RNA. When adenosine-5'-82phosphate (AM32P, obtained from the liver acid-soluble fraction be partially hepatectomized rats treated with inorganic sap) was incubated with the "cytoplasmic fraction" obtained by centrifuging the nuclei from a o.25 M sucrose homogenate of rat liver under conditions in which oxidative phosphorylation was maintained, radioactivity was incorporated * This work was supported in part by a research grant, C-24o9, from the National Cancer Institute, National Institutes of Health, Public Health Service, and in part by ~ grant from the Wisconsin Section of the American Cancer Society.
446
PRELIMINARY NOTES
VOL, 2 0 (1956)
into t h e R N A . Following isolation, t h e R N A was h y d r o l y z e d w i t h diesterase (prepared according to HURST AND BUTLERs f r o m dialyzed* s n a k e v e n o m ) , w h i c h cleaves t h e internucleotide p h o s p h o diester so as to give t h e 5 ' - m o n o n u c l e o t i d e s 5, t h e r a d i o a c t i v i t y w a s c o n c e n t r a t e d a l m o s t entirely in t h e 5 ' - A M P o b t a i n e d f r o m t h e h y d r o l y s a t e (Table I). T h u s t h e i n c u b a t e d nucleotide was incorporated into R N A w i t h o u t loss or r a n d o m i z a t i o n of its p h o s p h o r u s , a n d s h o w s clearly t h a t 5'-nucleotides are t h e a c t u a l i n t e r m e d i a t e s in R N A biosynthesis. R a t liver h o m o g e n i z e d in 0.25 M sucrose, a n d t h e s u p e r n a t a n t from 6oo × g c e n t r i f u g a t i o n was used. A RECOVERED RADIOACTIVITY FROM R N A o. 5 g e q u i v a l e n t of tissue was i n c u b a t e d in air at 3 °° C for 4 ° m i n u t e s in a t o t a l Diestemse hydrolysis Alkalinehydrolysis v o l u m e of 5.o ml including t h e follow(S') (~" + 39 ing c o n c e n t r a t i o n s of: O . o l M glutamate, o.oI M p y r u v a t e , o.004 M f u m a Adenylic acid 92 5-4 rate, o.oi M p h o s p h a t e buffer (pH 7.2), Cytidylic acid 0.6 87. 4 O . o l M m a g n e s i u m chloride, o.o331 Uridylic acid 0. 5 1.2 fructose, o.25.~I sucrose a n d ~ microG u a n y l i c acid o. 4 1.2 mole of AMz2p (specific activity, Inorganic phosphorus 6. I 4.8 6o8,ooo c.p.m./pM) 4. Four flasks were used a n d pooled. Acid-soluble fractions s a n d R N A 7were isolated, e n z y m i c a n d alkaline h y d r o l y s e s were carried o u t 2, a n d t h e nucleotides isolated b y g r a d i e n t elution chrom a t o g r a p h y on D o w e x - i 7. T h e uridylic acid was s e p a r a t e d from c o n t a m i n a t i n g inorganic p h o s p h a t e b y a d s o r p t i o n on charcoal, w a s h i n g w i t h water, a n d elution w i t h lO % pyridine. TABLE I
W h e n t h e s a m e s a m p l e of R N A was h y d r o l y z e d with alkali, which cleaves t h e internucleotide p h o s p h o diester to give a m i x t u r e of t h e m o n o n u c l e o t i d e - 2 ' a n d 3 ' - p h o s p h a t e s , t h e r a d i o a c t i v i t y was localized a l m o s t entirely in t h e cytidylic acid, a n d v e r y little w a s f o u n d in t h e o t h e r t h r e e nucleotides (Table I). T h e s e results s u g g e s t t h a t in t h e p a r t i c u l a r R N A t h a t was labeled u n d e r t h e s e conditions, ,adenosine is a d j a c e n t to cytidine, w i t h t h e radioactive p h o s p h a t e a t t a c h e d to t h e 5 ' - c a r b o n of t h e a d e n o s i n e a n d t h e 3 ' - c a r b o n of t h e cytidine. GOLDWASSER8 h a s v e r y r e c e n t l y reported t h e specific incorporation of 1*C-adenine-labeled A M P into t h e adenylic acid, o b t a i n e d b y alkaline h y d r o l y s i s of R N A , in a pigeon liver h o m o g e n a t e . However, in a similar e x p e r i m e n t u s i n g s ' P - l a b e l e d AMP, he f o u n d t h e label to be d i s t r i b u t e d a m o n g all four nucleotides r e s u l t i n g f r o m alkaline hydrolysis, w i t h t h e r a d i o a c t i v i t y in adenylic acid below t h e a v e r a g e a n d in uridylic acid, a b o v e t h e average. O u r results are also c o m p l e m e n t a r y to t h e e l e g a n t w o r k of GRUNBERG-MANAGO, ORTIZ, AND OCHOA9, w h o h a v e o b t a i n e d RNA-like p o l y n u c l e o t i d e s b y i n c u b a t i o n of n u c l e o s i d e - 5 ' - d i p h o s p h a t e s w i t h a polynucleotide p h o s p h o r y l a s e o b t a i n e d from Azotobacter vinelandii. I n our case, A D P m a y be t h e actual i n t e r m e d i a t e , w i t h t h e 32p of t h e A M P a c t u a l l y i n c o r p o r a t e d into t h e R N A since t h o s y s t e m p r o d u c e d A D P a n d A T P a n d t h e original inorganic p h o s p h o r u s w a s unlabeled. Similar e x p e r i m e n t s w i t h o t h e r nucleotides are b e i n g c o n t i n u e d a n d a m o r e detailed report will be p u b l i s h e d elsewhere.
~/IcArdle Memorial Laboratory, The Medical School, University o[ Wisconsin, Madison, Wis. (U.S.A.)
CHARLES HEIDELBERGER EBERHARD HARBERS** KENNETH C. LEIBMAN Y . TAKAGI
VAN R. POTTER I p. M. ROLL, H. WEINFELD AND G. B. BROWN, Biochim. Biophys. Acta, 13 (I954) 141. 2 K. C. LEIBMAN AND C. HEIDELBERGER, J. Biol. Chem., 216 (1955) 823. 3 V. R. POTTER et al., Biochim. Biophys. Acta, 2o (1956) 439. 4 R. O. HURST AND G. C. BUTLER, J. Biol. Chem., 193 (1951) 91. s W. E. COHN AND E. VOLKIN, J. Biol. Chem., 2o 3 (1953) 319. R. 13. HURLBERT, H. SCHMITZ, A. F. 13RUMM AND V. R. POTTER, J. Biol. Chem., 2o9 (1954) 23. R. 13. HURLBEET AND V. R. POTTER,J. Biol. Chem., I95 (I955) 257. s E. GOLDWASSE.R, J. Am. Chem. Soc., 77 (1955) 6o83. 9 M. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Science, 122 (1955) 9o7. Received March ist, 1956 * W e are g r e a t l y i n d e b t e d to Prof. EUGENE KENNEDY for t h e s u g g e s t i o n of dialyzing t h e crude v e n o m . ** F u l b r i g h t R e s e a r c h Fellow f r o m t h e U n i v e r s i t y of G 6 t t i n g e n , G e r m a n y .