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 properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum.

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca2+-ATPase and Mg2+-ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave Km and V values of 17.3 muM and 1.25 mumol Pi/min per mg, and 2.4 muM and 0.12 mumol Pi/min per mg, respectively. Adenylyl imidodiphosphate, a beta-gamma-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with Ki values equal to 350 and 12 muM in the presence of Ca2+ and Mg2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate (t1/2 = 24 h) by the ATP analog, 6,6'-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate (t1/2 approximately 10 min) skeletal and cardiac myosins rapidly by reacting with key cysteines.     

Purification of a myosin phosphatase from bovine aortic smooth muscle

A myosin phosphatase has been purified to homogeneity from bovine aortic smooth muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme eluted from nondenaturing gels revealed two subunits (Mr = 67,000 and 38,000). Densitometric scans of the subunits indicated a molar ratio of 1:1. Several phosphoproteins were substrates for the phosphatase including histone II-A, isolated 20,000-dalton smooth muscle myosin light chains, phosphorylase a, and smooth muscle myosin. In the presence of 0.25 M NaCl and a substrate concentration of 2 microM, myosin was preferentially dephosphorylated. The specific activity of the phosphatase for myosin at a concentration of 10 microM was found to be 5 mumol/mg/min. The phosphatase required Mn2+ or Co2+ ions for activity. Mg2+, Ca2+, or Mg-ATP would not substitute for Mn2+ or Co2+ at equimolar concentrations. This phosphatase may play an important role in regulating actin-myosin interaction in smooth muscle by serving to dephosphorylate myosin.     

The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Expression and characterization of recombinant thermostable alkaline phosphatase from a novel thermophilic bacterium Thermus thermophilus XM

A gene （tap） encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co^2＋, Fe^2＋, Mg^2＋, or Mn^2＋ but was strongly inhibited by 2.0 mM Fe^2＋. Under optimal conditions, the Michaelis constant （Kin） for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.     

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.     

Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].     

Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg.mL-1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0-8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.     

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: comparison with phosphoenolpyruvate phosphatase

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Ca2(+)-dependent protein phosphatase which dephosphorylates regulatory light chain-a in scallop smooth muscle myosin

Ca2(+)-dependent protein phosphatase was purified from scallop adductor smooth muscle by a combination of DEAE-Toyoperal 650S ion exchange chromatographies and gel filtration on Sephacryl S-300. The phosphatase consisted of two subunits having molecular weights of 60 and 19 kDa. Phosphorylated regulatory light chain-a (RLC-a) was dephosphorylated by this phosphatase both in free and bound states in myosin prepared from the opaque portion of scallop smooth muscle (opaque myosin). The dephosphorylation was activated by Ca2+. The half maximal activation was a 1 microM free Ca2+ in the presence of calmodulin and 7 microM free Ca2+ in the absence of calmodulin. Opaque myosin phosphorylated at the heavy chain was not dephosphorylated with this phosphatase. p-Nitrophenyl phosphate was dephosphorylated. In addition to Ca2+, the phosphatase activity for RLC-a was activated by Mn2+, while p-nitrophenylphosphatase activity was activated by Mg2+ more strongly than by Mn2+. The pH-activity curves showed a maximum at pH 7 in the presence of Mn2+, but at around pH 8 in the presence of Mg2+. This phosphatase is similar to phosphatase 2B or calcineurin. The possible regulatory function of this phosphatase in scallop catch muscle is discussed.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations

Preparations of sarcoplasmic reticulum vesicles, obtained according to the method of Eletr and Inesi (Biochim. Biophys. Acta (1972) 282, 174), contained both Mg2(+)-ATPase and Ca2+, Mg2(+)-ATPase activity. The two enzymes were solubilized by a mixture of digitonin and lysophosphatidylcholine and separated on a DEAE-cellulose column eluted with a discontinuous gradient of NaCl. The Mg2(+)-ATPase activity was eluted with 0.43 M NaCl. The Ca2+,Mg2(+)-ATPase was obtained by increasing the NaCl concentration of the elution medium to 0.40 M. The fraction eluted with 0.043 M NaCl was insensitive to micromolar concentrations of calcium, resistant to oligomycin, ouabain, orthovanadate and thiocyanate, and was inhibited by low concentrations of Triton X-100. The enzyme showed a single apparent Km for MgATP in the range of 0.2 mM and a Vm of 2.9 mumol Pi.min-1.mg-1 protein. Activity was maximal over a broad peak between pH 6.0-8.0. Hydrolysis of ATP was unaffected by dimethylsulfoxide concentrations up to 20% (v/v) and was inhibited at higher concentrations. The enzyme was not phosphorylated by either 32Pi or [gamma-32P]ATP at significant levels when compared with the Ca2+,Mg2(+)-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg2(+)-ATPase was distinctly different from that of the Ca2+,Mg2(+)-ATPase under the same conditions. The fraction eluted from the DEAE-cellulose column was subjected to electrophoresis under non-denaturing conditions. Only one band with Mg2(+)-ATPase activity was detected. The Mg2(+)-ATPase migrated much slower than the Ca2+,Mg2(+)-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.