Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Purification and Properties of a Glycoprotein Acid Phosphatase from Candida albicans

An acid phosphomonoesterase was purified 87-fold with a 4% recovery from disintegrated cells of Candida albicans by four stages of column chromatography. The purified enzyme was homogeneous by ultracentrifugal, electrophoretic, and immunological analyses. The fully corrected sedimentation coefficient, s(20,w), was calculated to be 5.51s. Molecular weight estimated from ultracentrifugal data was 124.3 x 10(3), from gel chromatography was 115 x 10(3), and from acrylamide gel electrophoretic data was 131 x 10(3). Buoyant density in sucrose was 1.15 g/cm(3). The enzyme was a mannoprotein with a hexose to protein ratio of 7: 1. The Michaelis constant of the enzyme was 3.3 x 10(-4) M for p-nitrophenyl phosphate as substrate, and the pH optimum was 4.5. The enzyme was competitively inhibited by inorganic phosphate (K(i) = 10(-4) M) and by arsenate (K(i) = 0.5 x 10(-4) M). A wide range of inorganic cations and anions did not affect enzyme activity, but Hg(2+), Cd(2+), and Cu(2+) were inhibitory. F(-) was also inhibitory at low concentrations, but the effect was reversed at higher concentrations. Phosphatase activity was completely destroyed by exposure of the enzyme to 70 C for 12 min, but was destroyed only slowly by proteolytic hydrolysis. The purified glycoprotein enzyme gave a line of identity with the "b" antigen of crude C. albicans homogenates in immunodiffusion and immunoelectrophoresis tests with sera from rabbits inoculated with intact C. albicans cells and from humans with proven candidiasis. Preliminary evidence suggests that the mannan and not the protein portion of the enzyme molecule is responsible for this antigenicity.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Isolation of transketolase from human erythrocytes

Transketolase was isolated from human red blood cells with over 6,200 fold purification by a new method. The stepwise procedure for the isolation of the enzyme from erythrocyte hemolysate included the use of ethanol/chloroform precipitation, chromatography on hydroxyapatite and finally, affinity adsorption on carboxymethyl-cellulose. The molecular weight of erythrocyte transketolase, as determined by polyacrylamide gel electrophoresis, appeared to be about 140,000. The pH optimum for activity was between 7.6 and 7.8 and the optimum temperature for activity was 50 degrees C. The Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 2.0 x 10(-4) M, 3.2 x 10(-4) M and 2.0 x 10(-3) M, respectively.     

Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.

The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.     

Purification of 5 beta-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.     

Adenosine deaminase: physical and chemical properties of partially purified mitochondrial and cytosol enzyme from rat liver.

Adenosine deaminase (ADA) was partially purified 486- and 994-fold from rat liver mitochondria and cytosol, respectively. Relative molecular mass of the enzymes from both fractions was 34,000. Km for adenosine and 2'-deoxy-adenosine were 3.08 x 10(-5) M and 3.03 x 10(-5) M for mitochondrial ADA and 3.12 x 10(-5) M and 2.87 x 10(-5) M for cytosolic ADA. The enzyme from both subcellular fractions had the maximum activity at pH 7.5-8.0, and pI 5.2 and 4.2 for mitochondrial and cytosolic enzyme, respectively. The enzyme was inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine and 2'-deoxycoformycin with Ki 4.4 x 10(-7) M and 3.2 x 10(-7) M for mitochondrial ADA and 4.9 x 10(-7) M 2.8 x 10(-7) M for cytosolic ADA. Among the natural nucleoside and deoxynucleotide derivatives tested, deoxy-GTP and UTP inhibited only cytosolic adenosine deaminase by 60% and 40%, respectively.     

Dimerization of ferulic and caffeic acids by purified peroxidase isolated from Bupleurum salicifolium callus culture

A novel peroxidase that catalyses the transformation of caffeic acid and ferulic acid via oxidative coupling was purified from callus cultures of Bupleurum salicifolium petioles. The enzyme, which was purified over 2,900-fold, is a glycoprotein with a molecular weight of 38,000, determined by SDS/PAGE and gel filtration. The K(m) values obtained were 2.4x10(-4) M for caffeic and 2.6x10(-4) M for ferulic acid, while the K(m) values for H2O2 with caffeic acid was 4x10(-5) M and for H2O2 with ferulic acid was 4.8x10(-4) M. The purified peroxidase exhibits lower activity with typical peroxidase substrates (guaiacol and pyrogallol) than it does with caffeic and ferulic acids, but does not exhibit any activity with other phenylpropanoids tested (cinnamic acid, coumaric acid, and 3,4-dimethoxycinnamic acid).     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Sulfite oxidase from Merluccius productus

Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was purified 482-fold from liver of the Pacific hake Merluccius productus. The molecular weight of the enzyme was found to be 120 000 by gel exclusion chromatography on Sephadex G-100. Electrophoretic analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gel revealed that the enzyme was composed of two subunits whose molecular weight was estimated to be 60 000. The pH optimum of the enzyme was 8.7; Ks for sulfite, 2.5 x 10(-5) M; and that for cytochrome c, 3.6 x 10(-7) M. The enzyme elicited an EPR signal at g = 1.97 characteristic of pentavalent molybdenum. Colorimetric analysis also disclosed that the enzyme contained 2 mol each of heme and molybdenum per mol of protein. This fish liver homogenate in isotonic sucrose solution was fractionated by differential centrifugation into nuclei, mitochondria, microsomes and supernatant (100 000 X g). The major portion of sulfite oxidase activity was found in mitochondria. The sulfite oxidase activity was markedly high in liver and kidney, as compared with that in heart, spleen, muscle, gill and eye.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Purification and activation of brain sulfotransferase.

Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.     

D-(+)-lactate dehydrogenase from Lactobacillus murinus

D-(+)-Lactate dehydrogenase from Lactobacillus murinus was purified 670-fold. The Mr was 140,000 as determined by gel filtration. Maximum enzymatic activity was observed at 25 degrees C and pH 6.0 in 200 mM Na2KPO4 buffer. When the temperature was increased from 60 to 65 degrees C, the enzyme was completely inactive in 5 min. The apparent Km for pyruvate and NADH were 4.7 x 10(-4) and 1 x 10(-5) M, respectively. Pyruvate analogs such as oxalate, oxamate, 2-oxobutyrate, and malonate acted as a competitive inhibitors. L-Lactate and L-malate were noncompetitive inhibitors.     

Hydroxylating activity of frog epidermis tyrosinase.

Trypsin activated in a similar way both the tyrosine hydroxylase and the dopa-oxidasa activities of frog epidermis tyrosinase. Several electron donors reduced or eliminated the lag period for the hydroxylating enzyme. 4 x 10(-5) M dopa was particularly effective, but without affecting the stationary activity after lag period. Tyrosine hydroxylase had KM = 2.6 X 10(-3) M for tyrosine and 2 x 10(-3) M dopa was a competitive inhibitor with Ki = 5 x 10(-4) M. The enzyme was inactivated during its actuation. Data on thermal denaturation were similar to other obtained from dopa oxidase. Our results tend to confirm our previous hypothesis that the activatory process of the enzyme is accompanied by a spatial unfolding of the enzyme molecule.     

Cadmium inhibition of a structural wheat peroxidase

The major peroxidase from 15-day-old wheat plants was purified to homogeneity by FPLC ion exchange and molecular exclusion chromatography. It consists of a single polypeptide of M(r) 37,500 according to gel filtration and SDS-PAGE and has a pI of 7.0. Kinetics of pyrogallol peroxidation showed that the enzyme follows the accepted mechanism for peroxidase, with kinetic constants k(1) =4.4x10(6) M(-1) s(-1) and k(3) =8.6x10(5) M(-1) s(-1). The effect of different metal ions was assayed on peroxidase activity. None of the ions used had any effect on enzyme activity, except for Cd(II), which was an inhibitor. This was an unexpected and novel finding for a peroxidase. The kinetics of pyrogallol peroxidation at different concentrations of Cd(II) have been studied and a mechanism for Cd(II) inhibition proposed. The results obtained could explain, in part, cadmium-induced oxidative stress.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.