CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Purification and properties of phosphoglycerate phosphomutase from spores and cells of Bacillus megaterium.

Phosphoglycerate phosphomutase has been purified to homogeneity from vegetative cells and germinated spores of Bacillus megaterium, and the spore and cell enzymes appear identical. The enzyme is a monomer of molecular weight 61,000. The compound 2,3-diphosphoglyceric acid is not required for activity, but the enzyme has an absolute and specific requirement for Mn2+. The enzyme is inhibited by ethylenediaminetetraacetate and sulfhydryl reagents, has a pH optimum of about 8.0, and has Km values for 3-phosphoglyceric acid and Mn2+ of 5 x 10(-4) and 4 x 10(-5) M, respectively.     

Purification and characterization of cytidine 5''-triphosphate:cytidine 5''-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca2+ or Mg2+ and was not affected by F-actin and ouabain. Vmax was 2.8 and 10.0 mumol Pi/mg protein per h for Ca2+- and Mg2+-dependent activity, respectively, and the corresponding Km was 4.8 X 10(-4) M and 3.2 X 10(-4) M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Phosphotyrosine phosphatase: a novel phosphatase specific for phosphotyrosine, 2'-AMP and p-nitrophenylphosphate in rat brain

A unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogenous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000. The molecular weight of the native enzyme was 45,000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8 X 10(-4) M, 1.4 X 10(-4) M, and 1.7 X 10(-4) M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8 X 10(-4) M and 2.0 X 10(-4) M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L(+)tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.     

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.     

Purification and properties of two deoxyribonucleases of Pseudomonas aeruginosa.

A survey of the major deoxyribonucleases in Pseudomonas aeruginosa strain PAO was undertaken. Two activities predominated in Brij-58 lysates of this organism. These have been purified from contaminating nuclease activities, and some of their properties have been elucidated. The first was a nuclease that degraded heat-denatured deoxyribonucleic acid (DNA) to mono- and dinucleotides. The activity of this enzyme was confined to single-stranded DNA, and 100% of the substrate was hydrolyzed to acid-soluble material. The Mg2+ optimum is low (1 to 3mM), and the molecular weight is 6 X 10(4). The second predominant activity was an adenosine 5'-triphosphate (ATP)-dependent deoxyribonuclease. This enzyme had an absolute dependence on the presence of ATP Mg2+ concentrations of approximately 10 mM. Five moles of ATP was consumed for each mole of phosphodiester bonds cleaved. The acid-soluble products of the reaction consisted of short oligonucleotides from one to six bases in length. Only 50% of the double-stranded DNA was rendered acid soluble in a limit digest. The molecular weight of this enzyme is 3 X 10(5). The observation of these enzymes in P. aeruginosa is consistent with the possibility that recombinational pathways similar to those of Escherichia coli are operating in this organism.     

Purification and properties of myo-inositol-1-phosphatase from rat brain

myo-Inositol-1-phosphatase [EC 3.1.3.25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29,000. The molecular weight of the native enzyme was 55,000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6. The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, beta-glycerophosphate, and alpha-glycerophosphate; the ratio of the reaction rates was 100 : 84 : 73 : 64 : 32. The Km values for inositol-1-phosphate, 2'-AMP, and beta-glycerophosphate were 1.2 X 10(-4) M, 1.9 X 10(-4) M, and 7.7 X 10(-4) M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with Ki values of 3 microM and 20 microM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mM. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions.     

Isolation and purification of restriction endonuclease SacI from Streptomyces achromogenes ATCC 12767

A procedure for isolation and purification of restriction endonuclease Sac I from Streptomyces achromogenes ATCC 12767 is proposed. It allows to obtain an electrophoretically homogeneous enzyme preparation with the purification degree 1097 and the enzyme yield by activity 3.7%. The molecular weight of SacI was found to be 52,000 +/- 5,000 D, and isoelectric point 6.2. The enzyme consists of two subunits, which was found by polyacrylamide gel electrophoresis under denaturing conditions. Km and Vmax values were determined for the enzymatic reaction; they are equal to 4.6 X 10(-9) M and 9.19 X 10(-10) M/min, respectively.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.     

Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.     

Cadmium-binding component in Escherichia coli during accommodation to low levels of this ion.

An inducible cadmium-binding protein was isolated from Escherichia coli cells accommodated to 3 X 10(-6) M Cd2+ but not from normal or unaccommodated cells. Sephadex G-100, metal chelate affinity chromatography, and disc gel electrophoresis were used in the purification procedure. The molecular weight of the Cd2+-binding protein was estimated to be about 39,000 by Sephadex G-100 chromatography, making it different from the conventional, much smaller metallothionein.     

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L.

An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000.     

Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum.

A protein kinase which phosphorylated histone and protamine was partially purified from bovine cerebellum. Casein and phosvitin were inert as substrates. The enzyme did not require any cyclic nucleotide. A sulfhydryl compound such as 2-mercaptoethanol, glutathione, or cysteine was necessary for the reaction. The optimum pH was 8.5 to 9.0 Km values for ATP and whole histone were 3.3 X 10(-6) M and 150 microgram/ml, respectively. The optimum concentration of Mg2+ varied with histone fractions employed; with H2B histone as substrate the enzyme was most active at 50 to 100 nM Mg2", whereas with H1 and H2A histones the maximum activity was observed at 5 to 10 mM Mg2+ and with H3 and H4 histones the enzyme was active over a range of 5 to 75 mM Mg2+. The enzyme phosphorylated Ser-32 and Ser-36 in H2B histone and Ser-38 in H1 histone, although the reaction with Ser-36 in H2B histone was very slow. The molecular weight was 6.4 X 10(4). The sedimentation coefficient and Stokes radium were about 4.5 and 29 A, respectively. The enzyme showed heterogeneity upon isoelectrofocusing electrophoresis with isoelectric points of 5.6, 6.0, and 6.6. The enzyme was not inhibited by protein inhibitor nor by the regulatory subunit of cyclic AMP-dependent protein kinase. Preliminary analysis suggested that the enzyme was produced from its precursor protein by a limited proteolytic reaction.     

Soluble cAMP-independent protein kinase from human spleen

A protein kinase (EC 2.7.1.37) was purified 2000-fold, from the soluble protein fraction of human spleen cells, using ion-exchange chromatography, ammonium sulfate fractionation, and gel filtration. This rapid procedure yielded 30% of the initial activity and an enzyme preparation with specific activity of 62 nmol min-1 mg-1 of protein. On the basis of disc gel electrophoresis in sodium dodecyl sulfate acrylamide gels and isoelectric focusing the enzyme preparation appears homogeneous and to consist of one polypeptide with a molecular weight of 43,000 and having a pI of 7.1. The purified enzyme activity is cyclic AMP and cGMP independent phosphorylates both alpha-casein and phosvitin, and uses Mg2+ ATP and Mg2+ GTP as phosphate donors, exhibiting an apparent Km of 2.0 and 6.6 X 10(-5)m, respectively. Furthermore, the enzyme activity is strongly inhibited by heparin (K50 = 0.1 micrograms/ml). These catalytic properties are characteristic of the enzyme casein kinase II, as described in several eukaryotic cells.