- Email: [email protected]
[24] Poly(ADP-ribose) synthetase from rat liver nuclei
154
NICOTINIC ACID; ANALOGS AND COENZYMES
[24]
ity of the same extent as a microsomal enzyme. 1° NADP, deamino-NAD, NMN, and thioNAD almost completely inhibit the enzyme at a 5 m M concentration. lo S. Green and A. Dobrjansky, J. Biol. Chem. 240, 2574 (1965).
[24] Poly(ADP-Ribose) Synthetase from Rat Liver Nuclei By
HIROTO OKAYAMA, KUNIHIRO U E D A a n d OSAMU HAYAISHI
nNAD + X -* (ADP-ribose)n--X + nNicotinamide + n H ÷ (X = a macromolecular acceptor)
Poly(ADP-ribose) synthetase, a chromatin-bound enzyme, polymerizes the ADP-ribose moiety of NAD with the concomitant release of nicotinamide. 1-3 The product, poly(ADP-ribose), is a linear polymer of ADP-ribosyl units linked by glycosidic ribosyl-(l"--~2')-ribose bonds. 1-3 In isolated nuclei, this product is synthesized in a form covalently bound to various nuclear proteins. 2"3 Histones HI and H2B are the major acceptors. 4-~ The highly purified synthetase from rat live# catalyzes the formation of poly(ADP-ribose) bound to an endogenous acceptor copurifying with the enzyme as well as the polymer bound to histones. 9 The purified enzyme also elongates the preexisting ADP-ribose chains chemically or enzymically attached to histone. 1°'11 In this chapter, purification and properties of the rat liver enzyme are described. 1 See this series, Y. Nishizuka, K. Ueda, and O. Hayaishi, Vol. 18 [130]. H. Hilz and P. Stone, Rev. Physiol. Biochem. Pharmacol. 76, 1 (1976). a O. Hayaishi and K. Ueda, Annu. Rev. Biochem. 46, 95 (1977). 4 y . Nishizuka, K. Ueda, T. Honjo, and O. Hayalshi, J. Biol. Chem. 243, 3765 (1968). P. T. Riquelme, L. O. Burzio, and S. S. Koide, Fed. Proc., Fed. Am. Soc. Exp. Biol. 36, 785 (1977). 8 N. C. W. Wong, G. G. Poirier, and G. H. Dixon, Eur. J. Biochem. 77, 11 (1977). 7 H. Okayama, K. Ueda, and O. Hayaishi, Proc. Natl. Acad. Sci. U.S.A. 75, 1111 (1978), a H. Okayama, C. M. Edson, M. Fukushima, K. Ueda, and O. Hayaishi, J. Biol. Chem. 252, 7000 (1977). 8 K. Ueda, O. Hayaishi, M. Kawaichi, N. Ogata, K. Ikai, J. Oka, and H. Okayama, in "Covalent and Non-Covalent Modulation of Protein Function" (D. Atkinson and C. F. Fox, eds.). Academic Press, New York, 1979. lo K. Ueda, M. Kawaichi, H. Okayama, and O. Hayaishi, J. Biol. Chem. 254, 679 (1979). n K. Ueda, H. Okayama, and O. Hayaishi, in "'Biochemical A s p e c t s o f Nutrition" (K. Yagi, ed.), p. 195. Jpn. Sci. Soc. Press, Tokyo, 1979.
METHODSIN ENZYMOLOGY,VOL. 66
Copyright© 1980by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0~12-181966-3
[24]
POLY(ADP-RIBOSE) SYNTHETASE
155
Assay
Principle. The synthesis of poly(ADP-ribose) is measured by determining the amount of radioactive ADP-ribose incorporated from [adenine-I~C]NAD into material insoluble in 20% trichloroacetic acid. Reagents Tris-HC1 buffer, 1 M, pH 8.0 MgCI2, 0.1 M Dithiothreitol, 0.1 M Calf thymus DNA (Sigma Chemical Co., St. Louis, Missouri; highly polymerized), 1 mg/ml Calf thymus whole histone (Sigma, Type IIA), 1 mg/ml [Adenine- U-14C]NAD, 1 m M (20,000 cpm/nmol) Heat-inactivated chromatin, 13 5 mg protein/ml H20, prepared by incubating rat liver chromatin at 50° for 20 min (NH4)2SO4, 4.0 M, pH 8.0 Trichloroacetic acid, 20%, 5% Procedures. (a) Assay 1 (standard assay). This is used to assay the purified enzyme. The reaction mixture contains 50/.d of Tris-HCl, 50/zl of MgC12, 5/zl of dithiothreitol, 10/zl of DNA, 20/zl of bistone, 5 p.1 of [14C]NAD, enzyme, and H20 in a total volume of 0.5 ml. The reaction is carried out at 37° for 10 min and stopped by the addition of 3 ml of icecold 20% trichloroacetic acid. The radioactivity incorporated into acidinsoluble material is collected on a Millipore filter (pore size, 0.45/zm), washed with 5 × 4 ml of 5% trichloroacetic acid, and quantified with a liquid scintillation spectrometer. When a high concentration of NAD is required, the assay conditions described by Shizuta et al. 1~ are recommended. (b) Assay 2. lz The samples at all steps of purification are assayed by this method; under these assay conditions, the interference by various salts of high concentration is suppressed and contaminating poly(ADPribose)-degrading enzymes are inhibited by (NH4)~SO4 .~4"1~The reaction mixture (0.5 ml) contains 0.2 ml of (NH4)2SO4 and 20/zl of heat-inactivated chromatin in place of DNA and histone. Other components of the reaction mixture are the same as those of Assay 1. The reaction is carried out at 37° for 1 hr, and the acid-insoluble radioactivity is determined as described above. 1~ See this volume [25 ]. is K. Yoshihara, Biochem. Biophys. Res. Commun. 47, 119 (1972). 14 K. Ueda, R. H. Reeder, T. Honjo, Y. Nishizuka, and O. Hayaishi, Biochem. Biophys. Res. Commun. 31,379 (1968). ,5 N. Miyakawa, K. Ueda, and O. Hayaishi, Biochem. Biophys. Res. Commun. 49, 239 (1972),,
156
NICOTINIC ACID: ANALOGS AND COENZYMES
[24]
Purification Procedure Typical purification is achieved on nuclei isolated from 400 g (wet weight) of rat liver. All operations are performed at 0-4 ° . All buffers used are adjusted to the specified pH values at 25°. Step 1. Nuclei Preparation. Wistar rats weighing 300-350 g are killed by decapitation and, after perfusion with 20 ml of ice-cold 0.9% NaCl, their livers are removed. Nuclei are prepared by the method of Chauveau et al. 16 and stored at -60 ° until use. Step 2. Chromatin Preparation. Nuclei isolated from 400 g of livers are successively washed with 50 ml each of 0.01 M Tris-HC1 (pH 7.4), 0.05 M Tris-HC1 (pH 7.8), 0.075 M NaCl containing 0.024 M ethylenediaminetetraacetate (pH 8.0), 0.05 M Tris-HC1 (pH 8.0), and 0.01 M TrisHC1 (pH 8.0) as described previously. 1'14 Step 3. KCI Extraction. Chromatin is stirred for 1 hr in 120 ml (final volume) of a 0.6 M KC1 solution containing 1 mM potassium phosphate (pH 7.2) and l mM dithiothreitol. After centrifugation at 105,000 g for l hr, the supernatant (100 ml) is obtained. Step 4. Hydroxyapatite Column Chromatography. After adjustment of the KCI concentration to 3 M by adding solid KC1, the extract is applied to a hydroxyapatite column (3.8 × 10 cm) preequilibrated with 1 mM potassium phosphate buffer (pH 7.2) containing 3 M KCI and 1 mM dithiothreitol. The column is washed with one bed volume of the same buffer and eluted with a linear gradient of potassium phosphate (pH 7.2) from l mM to 0.3 M, containing 3 M KCI and 1 mM dithiothreitol (total volume, 1000 ml). The flow rate is maintained at 100 ml/hr using a peristaltic pump. Fractions of 14 ml are collected. The enzyme activity is eluted in a single peak between 50 and 100 mM phosphate and is completely separated from DNA which comes out at about 0.18 M phosphate. Histones are eluted in a large peak of absorbance at 230 nm, ahead of the enzyme. To get a good resolution, a batch of hydroxyapatite with high capacity should be used, such as the preparation by the method of Mazin et al. 17 and that obtained from Clarkson Co. Some other commercially available preparations may give poor results. Step 5. (NH4)~S04 Fractionation. Hydroxyapatite fractions containing the activity are pooled and dialyzed overnight against 15 volumes of a 45% saturated (NH4)2SO4 solution containing 20 mM Tris-HCl (pH 7.5) and 1 mM dithiothreitol. The precipitate formed is removed by centrifugation at 10,000 g for 20 rain. The supernatant is adjusted to 75% saturation by adding solid (NH4)~SO4 and stirring for 30 min. The precip16 j. Chauveau, Y. Moul6, and C. Rouiller, Exp. Cell Res. 11, 317 (1956), 17 A. L. Mazin, G. E. Sulimova, and B. F. Vanyushin, Anal. Biochem. 61, 62 (1974).
[24]
157
POLY(ADP-RIBOSE) SYNTHETASE
itate is collected by centrifugation at 10,000 g for 20 min and dissolved in 2 ml of 50 mM Tris-HC1 (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The insoluble material is removed by centrifugation at 10,000 g for 10 min. Step 6. Sephadex G-150 Column Chromatography. The dissolved (NH4),,SO4 fraction is applied to a Sephadex G-150 column (2.5 x 80 cm) preequilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The column is eluted with the same buffer at a flow rate of 20 ml/hr. Fractions of 5 ml are collected. The enzyme activity is eluted in a single peak slightly after the void volume. Step 7. Phosphocellulose Column Chromatography. Active Sephadex fractions are pooled and directly applied to a phosphocellulose column (1.2 x 5 cm) equilibrated with 50 mM Tris-HC1 (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The column is washed with the same buffer and eluted with a linear gradient of 0.4-0.9 M KCI contained in the equilibration buffer (total volume, 80 ml) at a flow rate of 10 ml/hr. Fractions of 2.5 ml are collected. The activity is eluted around 0.65 M KC1 in good coincidence with the absorbance at 230 nm. The table summarizes the purification procedure. Properties
Purity and Stability. The final preparation of the enzyme does not contain any detectable amounts of nucleic acids. SDS-polyacrylamide gel electrophoresis of the enzyme gives a single band with a molecular weight of approximately 1.1 × 105. No stainable histone bands are de-
PURIFICATION OF POLY(ADP-RIBOSE) SYNTHETASE FROM RAT LIVER NUCLEI 8
Step Nuclei Chromatin KCI extract Hydroxyapatite 45-75% Ammonium sulfate Sephade× G-150 Phosphocellulose
Total protein (nag) 860 700 270 20 8.6 0.48 0.025
Total activity a (nmol/hr) 1150 3100 1780 730 300 320 170
a The activity was measured by Assay 2.
Specific activity a (nmol/hr/mg) 1.3 4.4 6.6 37 35 660 6900
Yield (%)
Purification (-fold)
100 269 155 63
1 3 5 28
26 28 15
27 507 5300
158
NICOTINIC ACID" ANALOGS AND COENZYMES
[24]
tectable, nor are poly(ADP-ribose) glycohydrolase and phosphodiesterase activities. The purified enzyme is stable at 0° for at least 2 weeks; repeated freezing and thawing causes a considerable loss of activity. In the presence of 30% glycerol, the enzyme is stable at -20 ° for 1-2 months. Requirements for Activity. The activity is absolutely dependent on the presence of DNA, and the addition of histone together with DNA stimulates the activity another 3- to 5-fold. Histone alone does not support the activity at all. A sulfhydryl reagent and magnesium ion are required for full activity. pH Optimum. In the presence of DNA alone or DNA plus histone, the optimal pH is approximately 9.0. Stimulation by histone is observed at any pHs. Kinetics. In the presence of DNA alone, the Km~value for NAD is 80 /zM, and the Vmax is 800 nmol/min/mg protein. In the presence of DNA plus histone, the Km is 25 /xM and the Vmax is almost doubled (1560 nmol/min/mg). Effects of Synthetic Polynucleotides. Poly(dA-dT) activates the enzyme 1.5 times more than calf thymus DNA. Single-stranded DNA, including poly(dA) and poly(dT), and natural or synthetic RNA are much less effective or almost ineffective. Effects of Histone Subfractions. All histone subfractions (H1, H2A, H2B, H3, and H4) activate the enzyme, at maximum, 3- to 5-fold. The maximal activation is obtained when histone is added in a ratio to DNA of 1 : 2 by weight. Excess amounts of any histone subfractions are highly inhibitory. Reaction Product. The average chain length of poly(ADP-ribose) synthesized under the standard assay conditions (Assay 1) varies from 1.5 to 10 ADP-ribosyl units, depending on the amount of an endogenous acceptor which copurifies with the enzyme. The polymers synthesized are mostly bound to an endogenous acceptor of unknown chemical nature, TM and partly to histone; the latter portion increases when higher concentrations of histone and DNA are used. 9
18 H. Okayama, C. M. Edson, M. Fukushima, and O. Hayaishi, in "'Poly(ADP-ribose) and ADP-ribosylation of Proteins" (H. Hilz, ed.), p. 1. de Gruyter, Berlin, 1976.
NICOTINIC ACID; ANALOGS AND COENZYMES
[24]
ity of the same extent as a microsomal enzyme. 1° NADP, deamino-NAD, NMN, and thioNAD almost completely inhibit the enzyme at a 5 m M concentration. lo S. Green and A. Dobrjansky, J. Biol. Chem. 240, 2574 (1965).
[24] Poly(ADP-Ribose) Synthetase from Rat Liver Nuclei By
HIROTO OKAYAMA, KUNIHIRO U E D A a n d OSAMU HAYAISHI
nNAD + X -* (ADP-ribose)n--X + nNicotinamide + n H ÷ (X = a macromolecular acceptor)
Poly(ADP-ribose) synthetase, a chromatin-bound enzyme, polymerizes the ADP-ribose moiety of NAD with the concomitant release of nicotinamide. 1-3 The product, poly(ADP-ribose), is a linear polymer of ADP-ribosyl units linked by glycosidic ribosyl-(l"--~2')-ribose bonds. 1-3 In isolated nuclei, this product is synthesized in a form covalently bound to various nuclear proteins. 2"3 Histones HI and H2B are the major acceptors. 4-~ The highly purified synthetase from rat live# catalyzes the formation of poly(ADP-ribose) bound to an endogenous acceptor copurifying with the enzyme as well as the polymer bound to histones. 9 The purified enzyme also elongates the preexisting ADP-ribose chains chemically or enzymically attached to histone. 1°'11 In this chapter, purification and properties of the rat liver enzyme are described. 1 See this series, Y. Nishizuka, K. Ueda, and O. Hayaishi, Vol. 18 [130]. H. Hilz and P. Stone, Rev. Physiol. Biochem. Pharmacol. 76, 1 (1976). a O. Hayaishi and K. Ueda, Annu. Rev. Biochem. 46, 95 (1977). 4 y . Nishizuka, K. Ueda, T. Honjo, and O. Hayalshi, J. Biol. Chem. 243, 3765 (1968). P. T. Riquelme, L. O. Burzio, and S. S. Koide, Fed. Proc., Fed. Am. Soc. Exp. Biol. 36, 785 (1977). 8 N. C. W. Wong, G. G. Poirier, and G. H. Dixon, Eur. J. Biochem. 77, 11 (1977). 7 H. Okayama, K. Ueda, and O. Hayaishi, Proc. Natl. Acad. Sci. U.S.A. 75, 1111 (1978), a H. Okayama, C. M. Edson, M. Fukushima, K. Ueda, and O. Hayaishi, J. Biol. Chem. 252, 7000 (1977). 8 K. Ueda, O. Hayaishi, M. Kawaichi, N. Ogata, K. Ikai, J. Oka, and H. Okayama, in "Covalent and Non-Covalent Modulation of Protein Function" (D. Atkinson and C. F. Fox, eds.). Academic Press, New York, 1979. lo K. Ueda, M. Kawaichi, H. Okayama, and O. Hayaishi, J. Biol. Chem. 254, 679 (1979). n K. Ueda, H. Okayama, and O. Hayaishi, in "'Biochemical A s p e c t s o f Nutrition" (K. Yagi, ed.), p. 195. Jpn. Sci. Soc. Press, Tokyo, 1979.
METHODSIN ENZYMOLOGY,VOL. 66
Copyright© 1980by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0~12-181966-3
[24]
POLY(ADP-RIBOSE) SYNTHETASE
155
Assay
Principle. The synthesis of poly(ADP-ribose) is measured by determining the amount of radioactive ADP-ribose incorporated from [adenine-I~C]NAD into material insoluble in 20% trichloroacetic acid. Reagents Tris-HC1 buffer, 1 M, pH 8.0 MgCI2, 0.1 M Dithiothreitol, 0.1 M Calf thymus DNA (Sigma Chemical Co., St. Louis, Missouri; highly polymerized), 1 mg/ml Calf thymus whole histone (Sigma, Type IIA), 1 mg/ml [Adenine- U-14C]NAD, 1 m M (20,000 cpm/nmol) Heat-inactivated chromatin, 13 5 mg protein/ml H20, prepared by incubating rat liver chromatin at 50° for 20 min (NH4)2SO4, 4.0 M, pH 8.0 Trichloroacetic acid, 20%, 5% Procedures. (a) Assay 1 (standard assay). This is used to assay the purified enzyme. The reaction mixture contains 50/.d of Tris-HCl, 50/zl of MgC12, 5/zl of dithiothreitol, 10/zl of DNA, 20/zl of bistone, 5 p.1 of [14C]NAD, enzyme, and H20 in a total volume of 0.5 ml. The reaction is carried out at 37° for 10 min and stopped by the addition of 3 ml of icecold 20% trichloroacetic acid. The radioactivity incorporated into acidinsoluble material is collected on a Millipore filter (pore size, 0.45/zm), washed with 5 × 4 ml of 5% trichloroacetic acid, and quantified with a liquid scintillation spectrometer. When a high concentration of NAD is required, the assay conditions described by Shizuta et al. 1~ are recommended. (b) Assay 2. lz The samples at all steps of purification are assayed by this method; under these assay conditions, the interference by various salts of high concentration is suppressed and contaminating poly(ADPribose)-degrading enzymes are inhibited by (NH4)~SO4 .~4"1~The reaction mixture (0.5 ml) contains 0.2 ml of (NH4)2SO4 and 20/zl of heat-inactivated chromatin in place of DNA and histone. Other components of the reaction mixture are the same as those of Assay 1. The reaction is carried out at 37° for 1 hr, and the acid-insoluble radioactivity is determined as described above. 1~ See this volume [25 ]. is K. Yoshihara, Biochem. Biophys. Res. Commun. 47, 119 (1972). 14 K. Ueda, R. H. Reeder, T. Honjo, Y. Nishizuka, and O. Hayaishi, Biochem. Biophys. Res. Commun. 31,379 (1968). ,5 N. Miyakawa, K. Ueda, and O. Hayaishi, Biochem. Biophys. Res. Commun. 49, 239 (1972),,
156
NICOTINIC ACID: ANALOGS AND COENZYMES
[24]
Purification Procedure Typical purification is achieved on nuclei isolated from 400 g (wet weight) of rat liver. All operations are performed at 0-4 ° . All buffers used are adjusted to the specified pH values at 25°. Step 1. Nuclei Preparation. Wistar rats weighing 300-350 g are killed by decapitation and, after perfusion with 20 ml of ice-cold 0.9% NaCl, their livers are removed. Nuclei are prepared by the method of Chauveau et al. 16 and stored at -60 ° until use. Step 2. Chromatin Preparation. Nuclei isolated from 400 g of livers are successively washed with 50 ml each of 0.01 M Tris-HC1 (pH 7.4), 0.05 M Tris-HC1 (pH 7.8), 0.075 M NaCl containing 0.024 M ethylenediaminetetraacetate (pH 8.0), 0.05 M Tris-HC1 (pH 8.0), and 0.01 M TrisHC1 (pH 8.0) as described previously. 1'14 Step 3. KCI Extraction. Chromatin is stirred for 1 hr in 120 ml (final volume) of a 0.6 M KC1 solution containing 1 mM potassium phosphate (pH 7.2) and l mM dithiothreitol. After centrifugation at 105,000 g for l hr, the supernatant (100 ml) is obtained. Step 4. Hydroxyapatite Column Chromatography. After adjustment of the KCI concentration to 3 M by adding solid KC1, the extract is applied to a hydroxyapatite column (3.8 × 10 cm) preequilibrated with 1 mM potassium phosphate buffer (pH 7.2) containing 3 M KCI and 1 mM dithiothreitol. The column is washed with one bed volume of the same buffer and eluted with a linear gradient of potassium phosphate (pH 7.2) from l mM to 0.3 M, containing 3 M KCI and 1 mM dithiothreitol (total volume, 1000 ml). The flow rate is maintained at 100 ml/hr using a peristaltic pump. Fractions of 14 ml are collected. The enzyme activity is eluted in a single peak between 50 and 100 mM phosphate and is completely separated from DNA which comes out at about 0.18 M phosphate. Histones are eluted in a large peak of absorbance at 230 nm, ahead of the enzyme. To get a good resolution, a batch of hydroxyapatite with high capacity should be used, such as the preparation by the method of Mazin et al. 17 and that obtained from Clarkson Co. Some other commercially available preparations may give poor results. Step 5. (NH4)~S04 Fractionation. Hydroxyapatite fractions containing the activity are pooled and dialyzed overnight against 15 volumes of a 45% saturated (NH4)2SO4 solution containing 20 mM Tris-HCl (pH 7.5) and 1 mM dithiothreitol. The precipitate formed is removed by centrifugation at 10,000 g for 20 rain. The supernatant is adjusted to 75% saturation by adding solid (NH4)~SO4 and stirring for 30 min. The precip16 j. Chauveau, Y. Moul6, and C. Rouiller, Exp. Cell Res. 11, 317 (1956), 17 A. L. Mazin, G. E. Sulimova, and B. F. Vanyushin, Anal. Biochem. 61, 62 (1974).
[24]
157
POLY(ADP-RIBOSE) SYNTHETASE
itate is collected by centrifugation at 10,000 g for 20 min and dissolved in 2 ml of 50 mM Tris-HC1 (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The insoluble material is removed by centrifugation at 10,000 g for 10 min. Step 6. Sephadex G-150 Column Chromatography. The dissolved (NH4),,SO4 fraction is applied to a Sephadex G-150 column (2.5 x 80 cm) preequilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The column is eluted with the same buffer at a flow rate of 20 ml/hr. Fractions of 5 ml are collected. The enzyme activity is eluted in a single peak slightly after the void volume. Step 7. Phosphocellulose Column Chromatography. Active Sephadex fractions are pooled and directly applied to a phosphocellulose column (1.2 x 5 cm) equilibrated with 50 mM Tris-HC1 (pH 7.5) containing 0.4 M KCI and 1 mM dithiothreitol. The column is washed with the same buffer and eluted with a linear gradient of 0.4-0.9 M KCI contained in the equilibration buffer (total volume, 80 ml) at a flow rate of 10 ml/hr. Fractions of 2.5 ml are collected. The activity is eluted around 0.65 M KC1 in good coincidence with the absorbance at 230 nm. The table summarizes the purification procedure. Properties
Purity and Stability. The final preparation of the enzyme does not contain any detectable amounts of nucleic acids. SDS-polyacrylamide gel electrophoresis of the enzyme gives a single band with a molecular weight of approximately 1.1 × 105. No stainable histone bands are de-
PURIFICATION OF POLY(ADP-RIBOSE) SYNTHETASE FROM RAT LIVER NUCLEI 8
Step Nuclei Chromatin KCI extract Hydroxyapatite 45-75% Ammonium sulfate Sephade× G-150 Phosphocellulose
Total protein (nag) 860 700 270 20 8.6 0.48 0.025
Total activity a (nmol/hr) 1150 3100 1780 730 300 320 170
a The activity was measured by Assay 2.
Specific activity a (nmol/hr/mg) 1.3 4.4 6.6 37 35 660 6900
Yield (%)
Purification (-fold)
100 269 155 63
1 3 5 28
26 28 15
27 507 5300
158
NICOTINIC ACID" ANALOGS AND COENZYMES
[24]
tectable, nor are poly(ADP-ribose) glycohydrolase and phosphodiesterase activities. The purified enzyme is stable at 0° for at least 2 weeks; repeated freezing and thawing causes a considerable loss of activity. In the presence of 30% glycerol, the enzyme is stable at -20 ° for 1-2 months. Requirements for Activity. The activity is absolutely dependent on the presence of DNA, and the addition of histone together with DNA stimulates the activity another 3- to 5-fold. Histone alone does not support the activity at all. A sulfhydryl reagent and magnesium ion are required for full activity. pH Optimum. In the presence of DNA alone or DNA plus histone, the optimal pH is approximately 9.0. Stimulation by histone is observed at any pHs. Kinetics. In the presence of DNA alone, the Km~value for NAD is 80 /zM, and the Vmax is 800 nmol/min/mg protein. In the presence of DNA plus histone, the Km is 25 /xM and the Vmax is almost doubled (1560 nmol/min/mg). Effects of Synthetic Polynucleotides. Poly(dA-dT) activates the enzyme 1.5 times more than calf thymus DNA. Single-stranded DNA, including poly(dA) and poly(dT), and natural or synthetic RNA are much less effective or almost ineffective. Effects of Histone Subfractions. All histone subfractions (H1, H2A, H2B, H3, and H4) activate the enzyme, at maximum, 3- to 5-fold. The maximal activation is obtained when histone is added in a ratio to DNA of 1 : 2 by weight. Excess amounts of any histone subfractions are highly inhibitory. Reaction Product. The average chain length of poly(ADP-ribose) synthesized under the standard assay conditions (Assay 1) varies from 1.5 to 10 ADP-ribosyl units, depending on the amount of an endogenous acceptor which copurifies with the enzyme. The polymers synthesized are mostly bound to an endogenous acceptor of unknown chemical nature, TM and partly to histone; the latter portion increases when higher concentrations of histone and DNA are used. 9
18 H. Okayama, C. M. Edson, M. Fukushima, and O. Hayaishi, in "'Poly(ADP-ribose) and ADP-ribosylation of Proteins" (H. Hilz, ed.), p. 1. de Gruyter, Berlin, 1976.