Actin kinase: a protein kinase that phosphorylates actin of fragmin-actin complex.

Actin of fragmin-actin complex is phosphorylated by an endogenous kinase from plasmodium of Physarum polycephalum. The phosphorylation abolishes the nucleation and capping activities of fragmin-actin complex. The kinase has been purified and termed actin kinase [Furuhashi, K. & Hatano, S. (1990) J. Cell Biol. 111, 1081-1087]. Enzymatic properties of the purified actin kinase were studied in detail. Actin kinase exhibited the highest activity under conditions physiological for the plasmodium (30 mM KCl, 6 mM MgCl2, pH 7.0). The Vmax and the Km of the enzyme for ATP were about 83 mumol/min/mg and 25 microM, respectively. The Km for fragmin-actin complex was 190 nM. The purified actin kinase phosphorylated actin of fragmin-actin complex at a constant rate regardless of Ca2+ concentration. Similarly, 2 microM cAMP, 2 microM cGMP, 2 micrograms/ml calmodulin in the presence of Ca2+ or 1 mM GTP showed no effect on the activity of the purified enzyme. Actin kinase did not phosphorylate histone H1, H2B, alpha-casein, or beta-casein, suggesting that actin kinase is a new kind of protein kinase which specifically phosphorylates actin of the fragmin-actin complex.     

Tritrichomonas foetus: purification and characterization of hydrogenosomal ATP:AMP phosphotransferase (adenylate kinase)

The hydrogenosomal enzyme ATP:AMP phosphotransferase (adenylate kinase) (EC 2.7.4.3) was purified to apparent homogeneity from the bovine parasite Tritrichomonas foetus. A fraction enriched for hydrogenosomes was obtained from cell homogenates which had been subjected to differential and isopycnic centrifugation. Adenylate kinase was solubilized in 50 mM Tris-HCl, pH 7.3, containing 0.8% Triton X-100, and purified by sequential Affi-Gel blue affinity chromatography and high-performance liquid chromatography gel filtration. The purified enzyme, a monomer of Mr 29,000, exhibited Km values of 100, 195, and 83 microM for ADP, ATP, and AMP, respectively. Substituting other mono-, di-, and trinucleotides for AMP, ADP, and ATP gave less than half the maximal activity. Full enzyme activity requires Mg2+, but Mn2+ and Co2+ yield half maximal activity. The enzyme has a broad optimal pH range between pH 6 and 9. The enzyme was competitively inhibited by P1,P5-di(adenosine-5')pentaphosphate, a specific adenylate kinase inhibitor: the Ki was 150 nM. The enzyme was also inhibited with 5,5'-dithiobis(2-nitrobenzoic acid), and this inhibition could be reversed by the addition of 2 mM dithiothreitol. T. foetus adenylate kinase has similar catalytic and physical properties to that of the biologically closely related human parasite Trichomonas vaginalis.     

Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus.

Acetohydroxy acid synthase (EC 4.1.3.18) of the archaebacterium Methanococcus aeolicus was purified 1,150-fold to homogeneity. The molecular weight of the purified enzyme was 125,000, and it contained only one type of subunit (M(r) = 58,000). The amino-terminal sequence had 46 to 57% similarity to those of the large subunits of the eubacterial anabolic enzymes and 37 to 43% similarity to those of the yeast and plant enzymes. The methanococcal enzyme had a pH optimum of 7.6. The pI, estimated by chromatofocusing, was 5.6. Activity required Mg2+ or Mn2+ ions, thiamine pyrophosphate, and a flavin. Flavin adenine dinucleotide, flavin mononucleotide, and riboflavin plus 10 mM phosphate all supported activity. However, activity was strongly inhibited by these flavins at 0.3 mM. The Michaelis constants for pyruvate, MgCl2, MnCl2, thiamine pyrophosphate, flavin adenine dinucleotide, and flavin mononucleotide were 6.8 mM, 0.3 mM, 0.16 mM, 1.6 microM, 0.4 microM, and 1.3 microM, respectively. In cell extracts, the enzyme was sensitive to O2 (half-life = 2.7 min with 5% O2 in the headspace), but the purified enzyme was less sensitive to O2 (half-life = 78.0 min with 20% O2). Reconstitution of the enzyme with flavin adenine dinucleotide increased the sensitivity to O2. Moreover, in the assay the homogeneous enzyme was rapidly inactivated by O2, and the concentration required for 50% inhibition (I50) was obtained with an atmosphere of 0.11% O2. The methanococcal enzyme has similarities to the eubacterial and eucaryotic enzymes, consistent with the ancient origin of the archaebacterial enzyme.     

The effect of Fe2+ on the activity of human plasma protein kinase

A Fe2+ stimulatory protein kinase in human plasma is demonstrated. This enzyme was partial purified from the 100,000 X g supernatant of human plasma by using (NH4)2SO4 fractionation. The activity of this protein kinase existed in 30-50% fraction. The effect of various compounds on the activity of this partial purified protein kinase was studied. The Fe2+ stimulates 5 to 10-fold of the protein kinase activity. The maximum concentration of Fe2+ to stimulate the activity of this plasma protein kinase was 2 mM. Double reciprocal plot for the stimulation showed that Fe2+ increased the Vm but not the Km for ATP. Other divalent compounds such as MgCl2, Mg(Oac)2, MnCl2 and BaCl2 were less effective.     

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa

The mitochondria of rat intestinal mucosa were found to have an enzymatic activity that converts radioactive glutamate to pyrroline-5-carboxylate (P5C) in the presence of ATP, NADPH, and MgCl2. The product of this enzyme was identified as P5C by the fact that it was converted to proline by chemical reduction with NaBH4 or by enzymatic reduction with NADH in the presence of purified yeast P5C reductase. The product was demonstrated to be P5C rather than pyrroline-2-carboxylate by thin layer chromatography. The presence of the activity in mitochondria prepared from intestinal mucosa of germ-free rats proved that this activity is of mammalian origin. Omission of either ATP, NADPH, or MgCl2 from the reaction mixture resulted in little or no activity. The optimal pH appeared to be about 7.0 under the conditions used. Substrate saturation curves in the presence of an ATP and an NADPH regeneration system gave apparent Km values of 2.5 mM for glutamate, 0.19 mM for ATP, and 6.5 microM for NADPH in the presence of 20 mM MgCl2. The mitochondrial preparation usually produced P5C at a rate of 1.2 to 1.6 nmol/mg/min at 20 degrees C when incubated with 1 mM glutamate, 3 mM ATP, 0.2 mM NADPH, and 20 mM MgCl2.     

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.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase.

Spermine in micromolar concentrations decreased the basal activity of a guanosine 3',5'-monophosphate (cGMP) phosphodiesterase from bovine brain but had no effect in the presence of Ca2+ plus the calcium-dependent regulatory protein (CDR) which increased the activity of the enzyme 4- to 6-fold. Similar effects of spermine were observed on the enzyme at several stages of purification. Spermidine and putrescine were also inhibitory but higher concentrations were required. In the absence of Ca2+ and CDR, the enzyme exhibited two apparent Km values for cGMP (2.5 and 20 microM) which were unaltered by spermine. In the presence of Ca2+ and CDR (when spermine had no effect on activity), a single Km (3.5 microM) was observed. Enzyme purified by chromatography on CDR-Sepharose was rapidly inactivated during incubation at 30 degrees C in 5 mM potassium phosphate buffer (pH 7.0) with EDTA and ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Spermine (20 microM) partially stabilized enzyme activity under these conditions, although it was somewhat less effective than 2 mM MgCl2. The inhibitory effects of spermine (or other polyamines) on basal phosphodiesterase activity, which can be overcome by Ca2+ and CDR, could be important in the regulation of cellular cyclic nucleotide content.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization

Adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 59,000-60,000 and is composed of two 30,000-dalton subunits. At 30 degrees C, pH 8.0 (0.1 M Tris-chloride buffer), 5.5 microM APS, 5 mM MgATP, 5 mM excess MgCl2, and "high" salt (70-150 mM (NH4)2SO4), the most highly purified preparation has a specific activity of 24.7 units X mg of protein-1 in the physiological direction of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation of APS kinase. APS kinase is subject to potent substrate inhibition by APS. In the absence of added salt, the initial velocity at 5 mM MgATP plus 5 mM Mg2+ is maximal at about 1 microM APS and half-maximal at 0.2 and 4.4 microM APS. In the presence of 200 mM NaCl or 70-150 mM (NH4)2SO4, the optimum APS concentration shifts to 4-6 microM APS; the half-maximal values shift to 1-1.3 and 21-27 microM APS. The steady state kinetics of the reaction were investigated using a continuous spectrophotometric assay. The families of reciprocal plots in the range 0.25-5 mM MgATP and 0.8-5.1 microM APS are linear and intersect on the horizontal axis. Appropriate replots yield KmMgATP = 1.5 mM, KmAPS = 1.4 microM, and Vmax, = 38.7 units X mg of protein-1. Excess APS is an uncompetitive inhibitor with respect to MgATP (K1APS = 23 microM). PAPS, the product of the forward reaction, is also uncompetitive with MgATP. PAPS is not competitive with APS. In the reverse direction, the plots have the characteristics of a rapid equilibrium ordered sequence with MgADP adding before PAPS. The kinetic constants are KmPAPS = 8 microM, KiMgADP = 560 microM, and Vmaxr = 0.16 units X mg of protein-1. Iso-PAPS (the 2'-phosphate isomer of PAPS) is competitive with PAPS and uncompetitive with respect to MgADP (Ki = 6 microM). APS kinase is inactivated by phenylglyoxal, suggesting the involvement of an essential argininyl residue. MgATP or MgADP at 10 Ki protect against inactivation. APS or PAPS at 600 and 80 Km, respectively, are ineffective alone, but provide nearly complete protection in the presence of 0.1 Ki of MgADP or MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of a distinct protamine kinase from the cytosol of bovine kidney cortex

A protamine kinase has been purified to apparent homogeneity from extracts of the cytosol of bovine kidney cortex. This protamine kinase exhibited an apparent Mr = 43,000 as estimated by gel permeation chromatography on Sephacryl S-200 and an apparent Mr = 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protamine kinase exhibited about 5% activity with casein, 8% with histone H2B, and less than 0.1% with histone H1, histone H4, glycogen synthase a from rabbit skeletal muscle, ovalbumin, bovine serum albumin, and phosvitin. The activity of the highly purified protamine kinase was unaffected by cyclic AMP (up to 0.1 mM), cyclic GMP (up to 0.1 mM), the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase (up to 100 micrograms/ml), heparin (up to 100 micrograms/ml), EGTA (up to 1 mM), Ca2+ (up to 1 mM), calmodulin (up to 0.5 microM) in the absence or presence of Ca2+ (0.05 mM), and phosphatidylserine (up to 40 micrograms/ml) and/or diolein (up to 1 microgram/ml) in the absence or presence of Ca2+ (up to 0.5 mM). Experiments in which extracts of kidney cytosol were incubated with [gamma-32P]ATP and MgCl2 revealed that the phosphorylation of numerous polypeptides was markedly increased in the presence of the purified protamine kinase. The results indicate that this protamine kinase of kidney cytosol is a novel protein kinase.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.     

Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle

A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.     

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.