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Acetylation of histones by pigeon liver enzymes
282
PRELIMINARY x()'l'ES
We wish to express our gratitude to Prof. N. KIMURA and Dr. S. KUBOTA who referred patient M.S. for study.
The Third Department of Internal Medicine, Osaka University Medical School, Osaka (Japan)
TOMOKAZU ~UZUKI AKIRA HAYASHI AKIRA SHIMIZU Y U I C H I YAMAMURA
I A. }{OSSI-FANELLI, E. ANTONINI AND :\. (~APUTO, in C. ]3. ANFINSEN, JR., .'~[. l.. ANSON', J. T. EDSALL AND F. M. t)tICHARDS, Advances in Prolei~z Chemist~2,,, Vol. 19, Academic Press, N~'w Y o r k - L o n d o n , 1964, p. 73. 2 T. SUZUKI, A. [-[AYASHI, Y. YAMAMURA, Y. ENOKI AND [. TYUMA, t'li~)chem, t3ioph~'.~, l?~s. Commun., 19 (1965) 691. 3 N. KIMURA, S. NISHINO, Y. NAWATA, F. MORI, S. KODAMA AND S. NAKAMURA, JaDa~i. lt~ar! .]., I (I96O) 456. 4 S. SHIBATA, T. MIYAJI, I. IUCHI, S. I'EI)A, I. TAKEI)A, N. I(IMURA ANI) S. [(OI)AM,, .lc/ct Haemalol. Japon., 25 (1962) 69o. 5 M. KIESE, H. KURZ AND C. SCHNEIDER, 14li~z. Wochschr., 34 (1956) 9576 K. MURA'WSKI, ~. CARTA, ~ . SORCINI, 1~. TENTORI, (_;. VIVALDI, E. ANTONINI, M. BRUNORI, J. WYMAN, E. B u c c l aNi) A. ROSSI-F.xNEULI, .4rch. Biochem. Biophys., l i t (i965) ~97. 7 N. HAYASHI, Y. MOTOKAWA AND G. KIKUCHI, .]. Biol. Chem., 241 11966 ) 79. 8 E. ANTONINI, E. BuccI, C. FRONTICELLI, J. \VYMAN AND :\. ROSSI-FaNELLL .]. Mol. Biol., I_, 11965) 3759 R. E. BENESCH, H. M. RANNEY, R. BENESCH AND (]. M. SMITH, .jr. Biol. Chem., 230 {i()(,l) 21)2().
Received June Ist, 1966 Biochim. Biophys. Acla, 127 (I966) 280-282
BBA 21 155 Acetylation of histones by pigeon liver enzymes The presence of acetyl end groups in thymus histones has been reported bv PHILIPS 1.
Recently
ALLFREY, FAULKNER AND MIRSKY 2
showed that histones
were
acetylated in nuclei isolated from calf thymus, and suggested that the acetylation of histones might influence the rate of R N A synthesis. These experiments prompted us to study the mechanism of enzymatic acetylation of histones. The present paper describes preliminary findings on the mechanism of acetylation of histones by pigeon liver enzymes and discusses the properties of acetyl groups accepted by the histone fractions. Acetylating enzymes were purified from pigeon liver by acetone fractionation according to the procedure of CHOWAND LIPMANNa, and the ability of acetone fractions to catalyze the incorporation of [14Clacetate into histones was examined 2. The results are shown in Table I. It was found that Fraction A-3o* definitely catalyzed the incorporation of [l*C]acetate into histones, although a significant uptake of [14C;acetate was observed by the enzyme itself. Furthermore, the incorporation of E14CJacetate into histones by Fraction A-3o was enhanced about two-to three-fold by the addition * Fractions A-3 o and A-6o were o b t a i n e d at 25 to 3 o~,o and 5 ° to 6o°/o acetone concentrations, respectively.
Biochim. Biophys. Acta, 127 11966) 282 284
283
PRELIMINARY NOTES TABLE I REQUIREMENTS FOR INCORPORATION OF [IIC]ACETATE INTO I-IISTONES
Complete s y s t e m : 50 mM Tris buffer (pH 7.4), 5 mM MgC12, 5 mM ATP, 6 mM GSH, 50 mM K F ' 20/~g CoA, lO 4 m/~moles [14C]acetate (9oo00 counts/min), 4 m g calf-thymus histones, o. 5 m g F r a c t i o n A-3o a n d 2.2 m g F r a c t i o n A-6o in i ml. The reaction m i x t u r e was incubated at 37 ° for 60 min. The reaction was s t o p p e d b y the addition of 18 % trichloroacetic acid. The precipitate was washed with 18% trichloroacetic acid, ethanol and m e t h a n o l - c h l o r o f o r m (3: I, v/v), and radioactivity was counted in a gas-flow counter.
System
Radioactivity (counts/min)
Acetate (mlamoles)
Complete Minus ATP Minus MgC12 Minus CoA Minus GSH Minus K F Minus F r a c t i o n A-6o Minus Fraction A-3o
147 ° 13 316 490 112o 17oo 41o 16
I. 7 O.Ol 5 0.36 0.56 1. 3 2.o 0.48 0.o2
of Fraction A-6o*. These results suggest that both activating and transferring enzymes are necessary for histone acetylation, as is the case for the acetylation of p-aminobenzoate s. In this acetylating system ATP, Mg ~+ and CoA were shown to be essential. In order to explore the chemical character of the acetyl groups incorporated into histones, hydroxylamine treatmenO, 5 of the acetylated histones was carried out. Thymus histones were labeled with [14C]acetate as described in Table I and the histones were extracted with 0.25 M HC1 and dialyzed against distilled water. The labeled histone was incubated with hydroxylamine (pH 6, final concn. 12 %) for 2 min, and then subjected to gel filtration on Sephadex G-5o (2 cm × 18 cm) to remove a Control -
-
~z
~
1 % ~
b Tr,eoted with hyclroxylomine for 5h _
1 2
Fr
i 6~
g E
O~
20
4O
60
80 1000 EfFluent (rnl)
20
40
60
80
1C
Fig. I. F r a c t i o n a t i o n of labeled histones on CM-cellulose column. 3 to 4 m g of labeled histones, after r e m o v i n g non-specific label as described in the text, was subjected to the second h y d r o x y l amine t r e a t m e n t a n d gel filtration. Then the protein was applied to CM-cellulose column (0.9 cm × 3 cm). An aliquot of each fraction was mixed with 15 ml of d i o x a n e - t o l u e n e - m e t h a n o l solution (375 : IOO : 25, v/v/v) containing o. 4 % 2,5-diphenyloxazole and o.oi % 1,4-bis-(5-phenyloxazolyl-2)-benzene, and the radioactivity was m e a s u r e d with a Packard liquid-scintillation spectrometer.
Biochim. Biophys. Acla, 127 (1966) 282-284
284
PRELIMINARY N OTF.S
non-specific label. The histone fraction was then c h r o m a t o g r a p h e d on a CM-eellulose c o l u m n b y stepwise elution 6. As shown in Fig. la, all the histone fractions were labeled. I t is n o t e w o r t h y t h a t F r a c t i o n I I I (arginine-rich fraction) had a considerably higher specific a c t i v i t y t h a n the other fractions. NARITA4 a n d PEIcLMANN~ reported t h a t the t r e a t m e n t of a c e t y l a t e d (formylated) protein with h y d r o x y l a m i n e resulted in the release of O-acetyl (O-formyl) groups from protein while N-acetvl groups were not released u n d e r the same condition. The labeled histone fraction was further t r e a t e d with h y d r o x y l a m i n e at 3 °° for 5 h as described b y NAI~ITA~, with s u b s e q u e n t gel filtration on Sephadex G-5o. This h y d r o x y l a m i n e - t r e a t e d histone was then chrom a t o g r a p h e d on a CM-cellulose column. As shown in Fig. Ib, the specific activit\' of F r a c t i o n I I I was reduced to about 5o % of the control value while the activities of F r a c t i o n s I a n d I I were v i r t u a l l y unchanged. F u r t h e r m o r e , a e e t y l h y d r o x a m a t e was identified b y paper c h r o m a t o g r a p h y : of the fraction released b y t r e a t m e n t with h y d r o x y l a m i n e for 5 h. These results suggest t h a t the arginine-rich fraction (Fraction IfI) of t h y m u s histones accepts acetate as labile acetyl groups (O-acetyl) a n d also as stable acetyl groups (N-acetyl), whereas lysine-rich (Fraction l) and slightly lysine-rich fraction (Fraction II) accept acetate only as stable aeetyl groups. MAteCHIs-M()I:Ri~X AXl) LIPMANNs have also reported t h a t acetate is transferred from acetyl-CoA to chicken hemoglobin b y an e n z y m e from chicken reticulocytes, although their enzyme did not catalyze the acetylation of c a l f - t h y m u s histones. The authors would like to t h a n k Professor K. NARITA for his suggestions and discussion on this work. Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) i 2 3 4 5 6 7
H . N OHARA
T. TAKAHASHI K. OGATA
D. M. P. PHILIPS, Biochem. ,J., 87 (I963) 258. V. G. ALLFREY, ]{. FAULKNERAND A. E. 3/~IRSKY,Proc. Nail. Acad. ,~ici. ~:..~'., 51 (t9b,t) 7,~,. T. C. CHOUAND F. LIPM*NN, J. Biol. Chem., 196 (I951) 89. K. NARITA,J. Am. Chem. Soc., 81 (1959) 1751. G. E. PERLMANN,J. Biol. Chem., 241 (1966) 153. E. VV. JOHNS, D. M. P. PHILIPS, P. SIMSONAND J. A. V. BUTLER, Biochem..]., 77 (~90o} 031. R. J. BLOCK,E. L. DURRUMAND G. ZWEIG,A manual of paper chromatography and paper electrophoresis, Part 1, Academic Press, New York, 2nd ed., 1958, p. 220. 7 R. J. BLOCK, E. L. DURRUMAND G. ZWEIG, A Manual of Paper Chromatography and Paper Alectrophoresis, Part 1, Academic Press, New York, 2nd ed., ~958, p. 220. 8 G. MARCHIS-MOURENAND F. LIPMANN,Proc. Natl. Acad. Sci. U.S., 53 (1965) i 147. Received J u l y 22nd, 1966 Biochim. Biophys. Aeta, I27 (1966) 282 284
PRELIMINARY x()'l'ES
We wish to express our gratitude to Prof. N. KIMURA and Dr. S. KUBOTA who referred patient M.S. for study.
The Third Department of Internal Medicine, Osaka University Medical School, Osaka (Japan)
TOMOKAZU ~UZUKI AKIRA HAYASHI AKIRA SHIMIZU Y U I C H I YAMAMURA
I A. }{OSSI-FANELLI, E. ANTONINI AND :\. (~APUTO, in C. ]3. ANFINSEN, JR., .'~[. l.. ANSON', J. T. EDSALL AND F. M. t)tICHARDS, Advances in Prolei~z Chemist~2,,, Vol. 19, Academic Press, N~'w Y o r k - L o n d o n , 1964, p. 73. 2 T. SUZUKI, A. [-[AYASHI, Y. YAMAMURA, Y. ENOKI AND [. TYUMA, t'li~)chem, t3ioph~'.~, l?~s. Commun., 19 (1965) 691. 3 N. KIMURA, S. NISHINO, Y. NAWATA, F. MORI, S. KODAMA AND S. NAKAMURA, JaDa~i. lt~ar! .]., I (I96O) 456. 4 S. SHIBATA, T. MIYAJI, I. IUCHI, S. I'EI)A, I. TAKEI)A, N. I(IMURA ANI) S. [(OI)AM,, .lc/ct Haemalol. Japon., 25 (1962) 69o. 5 M. KIESE, H. KURZ AND C. SCHNEIDER, 14li~z. Wochschr., 34 (1956) 9576 K. MURA'WSKI, ~. CARTA, ~ . SORCINI, 1~. TENTORI, (_;. VIVALDI, E. ANTONINI, M. BRUNORI, J. WYMAN, E. B u c c l aNi) A. ROSSI-F.xNEULI, .4rch. Biochem. Biophys., l i t (i965) ~97. 7 N. HAYASHI, Y. MOTOKAWA AND G. KIKUCHI, .]. Biol. Chem., 241 11966 ) 79. 8 E. ANTONINI, E. BuccI, C. FRONTICELLI, J. \VYMAN AND :\. ROSSI-FaNELLL .]. Mol. Biol., I_, 11965) 3759 R. E. BENESCH, H. M. RANNEY, R. BENESCH AND (]. M. SMITH, .jr. Biol. Chem., 230 {i()(,l) 21)2().
Received June Ist, 1966 Biochim. Biophys. Acla, 127 (I966) 280-282
BBA 21 155 Acetylation of histones by pigeon liver enzymes The presence of acetyl end groups in thymus histones has been reported bv PHILIPS 1.
Recently
ALLFREY, FAULKNER AND MIRSKY 2
showed that histones
were
acetylated in nuclei isolated from calf thymus, and suggested that the acetylation of histones might influence the rate of R N A synthesis. These experiments prompted us to study the mechanism of enzymatic acetylation of histones. The present paper describes preliminary findings on the mechanism of acetylation of histones by pigeon liver enzymes and discusses the properties of acetyl groups accepted by the histone fractions. Acetylating enzymes were purified from pigeon liver by acetone fractionation according to the procedure of CHOWAND LIPMANNa, and the ability of acetone fractions to catalyze the incorporation of [14Clacetate into histones was examined 2. The results are shown in Table I. It was found that Fraction A-3o* definitely catalyzed the incorporation of [l*C]acetate into histones, although a significant uptake of [14C;acetate was observed by the enzyme itself. Furthermore, the incorporation of E14CJacetate into histones by Fraction A-3o was enhanced about two-to three-fold by the addition * Fractions A-3 o and A-6o were o b t a i n e d at 25 to 3 o~,o and 5 ° to 6o°/o acetone concentrations, respectively.
Biochim. Biophys. Acta, 127 11966) 282 284
283
PRELIMINARY NOTES TABLE I REQUIREMENTS FOR INCORPORATION OF [IIC]ACETATE INTO I-IISTONES
Complete s y s t e m : 50 mM Tris buffer (pH 7.4), 5 mM MgC12, 5 mM ATP, 6 mM GSH, 50 mM K F ' 20/~g CoA, lO 4 m/~moles [14C]acetate (9oo00 counts/min), 4 m g calf-thymus histones, o. 5 m g F r a c t i o n A-3o a n d 2.2 m g F r a c t i o n A-6o in i ml. The reaction m i x t u r e was incubated at 37 ° for 60 min. The reaction was s t o p p e d b y the addition of 18 % trichloroacetic acid. The precipitate was washed with 18% trichloroacetic acid, ethanol and m e t h a n o l - c h l o r o f o r m (3: I, v/v), and radioactivity was counted in a gas-flow counter.
System
Radioactivity (counts/min)
Acetate (mlamoles)
Complete Minus ATP Minus MgC12 Minus CoA Minus GSH Minus K F Minus F r a c t i o n A-6o Minus Fraction A-3o
147 ° 13 316 490 112o 17oo 41o 16
I. 7 O.Ol 5 0.36 0.56 1. 3 2.o 0.48 0.o2
of Fraction A-6o*. These results suggest that both activating and transferring enzymes are necessary for histone acetylation, as is the case for the acetylation of p-aminobenzoate s. In this acetylating system ATP, Mg ~+ and CoA were shown to be essential. In order to explore the chemical character of the acetyl groups incorporated into histones, hydroxylamine treatmenO, 5 of the acetylated histones was carried out. Thymus histones were labeled with [14C]acetate as described in Table I and the histones were extracted with 0.25 M HC1 and dialyzed against distilled water. The labeled histone was incubated with hydroxylamine (pH 6, final concn. 12 %) for 2 min, and then subjected to gel filtration on Sephadex G-5o (2 cm × 18 cm) to remove a Control -
-
~z
~
1 % ~
b Tr,eoted with hyclroxylomine for 5h _
1 2
Fr
i 6~
g E
O~
20
4O
60
80 1000 EfFluent (rnl)
20
40
60
80
1C
Fig. I. F r a c t i o n a t i o n of labeled histones on CM-cellulose column. 3 to 4 m g of labeled histones, after r e m o v i n g non-specific label as described in the text, was subjected to the second h y d r o x y l amine t r e a t m e n t a n d gel filtration. Then the protein was applied to CM-cellulose column (0.9 cm × 3 cm). An aliquot of each fraction was mixed with 15 ml of d i o x a n e - t o l u e n e - m e t h a n o l solution (375 : IOO : 25, v/v/v) containing o. 4 % 2,5-diphenyloxazole and o.oi % 1,4-bis-(5-phenyloxazolyl-2)-benzene, and the radioactivity was m e a s u r e d with a Packard liquid-scintillation spectrometer.
Biochim. Biophys. Acla, 127 (1966) 282-284
284
PRELIMINARY N OTF.S
non-specific label. The histone fraction was then c h r o m a t o g r a p h e d on a CM-eellulose c o l u m n b y stepwise elution 6. As shown in Fig. la, all the histone fractions were labeled. I t is n o t e w o r t h y t h a t F r a c t i o n I I I (arginine-rich fraction) had a considerably higher specific a c t i v i t y t h a n the other fractions. NARITA4 a n d PEIcLMANN~ reported t h a t the t r e a t m e n t of a c e t y l a t e d (formylated) protein with h y d r o x y l a m i n e resulted in the release of O-acetyl (O-formyl) groups from protein while N-acetvl groups were not released u n d e r the same condition. The labeled histone fraction was further t r e a t e d with h y d r o x y l a m i n e at 3 °° for 5 h as described b y NAI~ITA~, with s u b s e q u e n t gel filtration on Sephadex G-5o. This h y d r o x y l a m i n e - t r e a t e d histone was then chrom a t o g r a p h e d on a CM-cellulose column. As shown in Fig. Ib, the specific activit\' of F r a c t i o n I I I was reduced to about 5o % of the control value while the activities of F r a c t i o n s I a n d I I were v i r t u a l l y unchanged. F u r t h e r m o r e , a e e t y l h y d r o x a m a t e was identified b y paper c h r o m a t o g r a p h y : of the fraction released b y t r e a t m e n t with h y d r o x y l a m i n e for 5 h. These results suggest t h a t the arginine-rich fraction (Fraction IfI) of t h y m u s histones accepts acetate as labile acetyl groups (O-acetyl) a n d also as stable acetyl groups (N-acetyl), whereas lysine-rich (Fraction l) and slightly lysine-rich fraction (Fraction II) accept acetate only as stable aeetyl groups. MAteCHIs-M()I:Ri~X AXl) LIPMANNs have also reported t h a t acetate is transferred from acetyl-CoA to chicken hemoglobin b y an e n z y m e from chicken reticulocytes, although their enzyme did not catalyze the acetylation of c a l f - t h y m u s histones. The authors would like to t h a n k Professor K. NARITA for his suggestions and discussion on this work. Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) i 2 3 4 5 6 7
H . N OHARA
T. TAKAHASHI K. OGATA
D. M. P. PHILIPS, Biochem. ,J., 87 (I963) 258. V. G. ALLFREY, ]{. FAULKNERAND A. E. 3/~IRSKY,Proc. Nail. Acad. ,~ici. ~:..~'., 51 (t9b,t) 7,~,. T. C. CHOUAND F. LIPM*NN, J. Biol. Chem., 196 (I951) 89. K. NARITA,J. Am. Chem. Soc., 81 (1959) 1751. G. E. PERLMANN,J. Biol. Chem., 241 (1966) 153. E. VV. JOHNS, D. M. P. PHILIPS, P. SIMSONAND J. A. V. BUTLER, Biochem..]., 77 (~90o} 031. R. J. BLOCK,E. L. DURRUMAND G. ZWEIG,A manual of paper chromatography and paper electrophoresis, Part 1, Academic Press, New York, 2nd ed., 1958, p. 220. 7 R. J. BLOCK, E. L. DURRUMAND G. ZWEIG, A Manual of Paper Chromatography and Paper Alectrophoresis, Part 1, Academic Press, New York, 2nd ed., ~958, p. 220. 8 G. MARCHIS-MOURENAND F. LIPMANN,Proc. Natl. Acad. Sci. U.S., 53 (1965) i 147. Received J u l y 22nd, 1966 Biochim. Biophys. Aeta, I27 (1966) 282 284