Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.     

Purification and characterization of a catalytic subunit of an adenosine 3':5'-monophosphate-dependent protein kinase from human erythrocyte membranes

Protein kinase [EC 2.7.1.37] of human erythrocyte membranes was solubilized with 0.5 M NaCl in 5 mM phosphate buffer, pH 6.7 at 4 degrees C and purified on a CM-Sephadex C-50 column, followed by affinity chromatography on a histone-Sepharose 4B column. The purified protein kinase gave a single band (molecular weight; 41,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 8.0 and a millimolar range of concentration of Mg2+ was required for its maximum activity. Histone and protamine were well phosphorylated by the protein kinase but casein and phosvitin were poor phosphate acceptors for the enzyme. The enzymic activity was not stimulated by cyclic AMP (cAMP). A cAMP-finding protein from human erythrocyte membranes inhibited the activity of the protein kinase, but the activity was restored with cAMP. A heat stable protein inhibitor from rabbit skeletal muscle also inhibited this enzyme. From these observations, this protein kinase seemed to be a catalytic subunit of the membrane bound cAMP-dependent protein kinase. This enzyme was strongly inhibited with Ca2+ in the presence of 1 mM MgCl2. Various sulfhydryl reagents and polyamines also had inhibitory activity on the protein kinase. Natural substrates of the enzyme were investigated using heat treated membranes and 0.5 M NaCl extracted membrane residues. Band 4.1, 4.2, and 4.5 proteins were phosphorylated but band 2 (spectrin) and band 3 proteins were poor substrates for this protein kinase.     

Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei.

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl(2) (10(-5) and 10(-4) M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 microM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10(-6)-10(-4) M), an inhibitor of Ca(2+)/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca(2+)/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 microM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II

Incubation of clathrin-coated vesicles with Mg2+-[gamma-32P]ATP results in the autophosphorylation of a 50-kDa polypeptide (pp50) (Pauloin, A., Bernier, I., and Jollès, P. (1982) Nature 298, 574-576). We describe here a second protein kinase that is associated with calf brain and liver coated vesicles. This kinase, which phosphorylates casein and phosvitin but not histone and protamine using either ATP or GTP, co-fractionates with coated vesicles as assayed by gel filtration, electrophoresis, and sedimentation. The enzyme can be extracted with 0.5 M Tris-HCl or 1 M NaCl, and can be separated from the pp50 kinase as well as the other major coat proteins. We identified this enzyme as casein kinase II based on physical and catalytic properties and by comparative studies with casein kinase II isolated from brain cytosol. It has a Stokes radius of 4.5 nm, a catalytic moiety of approximately 45 kDa, and labels a polypeptide of 26 kDa when the pure enzyme is assayed for autophosphorylation. Its activity is inhibited by heparin and not affected by cAMP, phospholipids, or calmodulin. This protein kinase preferentially phosphorylates clathrin beta-light chain. The phosphorylation is markedly stimulated by polylysine and inhibited by heparin. Isolated beta-light chain as well as beta-light chain in triskelions or in intact coated vesicles is phosphorylated. All of the phosphate (0.86 mol of Pi/mol of clathrin beta-light chain) is incorporated into phosphoserine.     

Autophosphorylation and autoactivation of spleen protein tyrosine kinase

Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration.     

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.     

Phosphorylation in vivo of red-muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress

That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

Purification and characterization of neutral trehalase from the yeast ABYS1 mutant

Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.     

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.     

Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity

A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.     

Nonactivated phosphorylase kinase is a phosphoprotein: differentiation of two classes of endogenous phosphoserine residues by phosphorus-31 nuclear magnetic resonance spectroscopy and phosphatase sensitivity

A standard preparation of phosphorylase kinase from rabbit skeletal muscle contains 2 mol of phosphoserine/mol of alpha beta gamma delta. This basal stoichiometry is not influenced by application of propranolol and insulin in vivo; these serine phosphates could not be hydrolyzed by phosphatases of the muscle extract or by alkaline phosphatases. When the enzyme is purified in the presence of the protein phosphatase inhibitor sodium fluoride, it contains either 1 or 3 additional mol of phosphoserine/mol of alpha beta gamma delta, termed phosphatase-sensitive phosphates. Both classes of phosphates yield in formic acid one single 31P NMR signal of a narrow line width (approximately 3 Hz) very similar in chemical shift to free phosphoserine. Phosphoserine is also identified by its chemical shift when dissolved in 8 M guanidinium chloride and by its electrophoretic mobility after acid hydrolysis. By self-phosphorylation of phosphorylase kinase, 14 additional mol of phosphate/mol of alpha beta gamma delta was incorporated, and all were identified as phosphoserine by 31P NMR spectroscopy. In native phosphorylase kinase, the 31P NMR signals of both the basal and the phosphatase-sensitive phosphates are substantially broadened and reduced in intensity, indicating strong interactions of the phosphate groups with the protein. The basal and phosphatase-sensitive phosphates give in 8 M guanidinium chloride a homogeneous NMR signal above pH 6; it splits into a doublet below pH 6 and into a triplet below pH 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.     

Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions

The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase.     

Identification and purification of a cytosolic phosphotyrosyl protein phosphatase from bovine spleen

A protein tyrosine kinase with an apparent Mr of 60,000 was highly purified from bovine spleen and used to phosphorylate poly(Glu, Tyr) (4:1) on tyrosine residues for the study of phosphotyrosyl protein phosphatases from this tissue. About 70% of the phosphotyrosyl protein phosphatase activity in extracts of bovine spleen was adsorbed on DEAE-Sepharose. Chromatography of the eluted phosphotyrosyl protein phosphatases on phosphocellulose indicated the presence of at least two species, one that did not bind to the phosphocellulose and a second species that did bind and was eluted at about 0.5 M NaCl. The phosphatase that did not bind to phosphocellulose was further purified by successive chromatography on poly(L-lysine)-Sepharose, L-tyrosine-agarose, poly(Glu,Tyr)-Sepharose, and Sephacryl S-200. The enzyme had an apparent Mr of 50,000 as estimated by gel filtration and 52,000 as estimated by NaDodSO4- polyacrylamide gel electrophoresis. The phosphatase exhibited a pH optimum of 6.5-7.0, was inhibited by Zn2+ and vanadate ions, and was stimulated by EDTA. Sodium fluoride and sodium pyrophosphate, inhibitors of phosphoseryl protein phosphatases, had no effect on the enzyme. Protein inhibitors of type 1 phosphoseryl/threonyl phosphatase were also ineffective.     

Role of protein phosphorylation in the maturation-induced activation of a myelin basic protein kinase from sea star oocytes

We have previously described the purification of a myelin basis protein (MBP) kinase from maturing sea star oocytes (Sanghera, J. S., Paddon, H. B., Bader, S. A., and Pelech, S. L. (1990) J. Biol. Chem. 265, 52-57). The ability of the purified 44-kDa protein to bind azido-ATP and undergo autophosphorylation on the serine residue implied that it is a protein kinase. Furthermore, partial amino acid sequence data has revealed that it is a novel protein kinase, which we have provisionally designated p44mpk. Autophosphorylation of p44mpk to 0.7 mol of phosphate/mol of enzyme was correlated with a modest (approximately 17%) increase in the MBP-phosphorylating activity of the kinase. Rabbit polyclonal antibody raised against purified p44mpk recognized on immunoblots the protein in highly purified preparations as well as crude oocyte extracts. The affinity-purified anti-p44mpk antibody could immunoprecipitate active kinase, but a subpopulation of the antibody also appeared to be inhibitory. Using this antibody, we have demonstrated that the up to 12-fold stimulation of the cytosolic MBP-phosphorylating activity of this kinase that occurs during sea star oocyte maturation is not due to an increase in the amount of enzyme protein, either from a redistribution within the oocyte or protein synthesis. A slight retardation of the migration of the activated p44mpk on sodium dodecyl sulfate-polyacrylamide gels and its tighter interaction with a MonoQ column is consistent with phosphorylation of the kinase during maturation. p44mpk underwent enhanced phosphorylation when oocytes prelabeled with [32P]orthophosphate were induced to mature with 1-methyladenine. The stimulated MBP-phosphorylating activity of p44mpk in cytosols from maturing oocytes was partly stabilized by the presence of the phosphatase inhibitor beta-glycerol phosphate. Furthermore, treatment of purified p44mpk with protein phosphatase 2A and alkaline phosphatase resulted in 56 and 86% decreases, respectively, in the activity of the kinase. Together, these findings strongly implicate a role for phosphorylation of p44mpk in its activation during sea star oocyte maturation.     

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.     

Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans

The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP.     

Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.