Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A density gradient-purified microsomal membrane preparation from rabbit fundic gastric mucosa was used for a detailed study of the K+-stimulated ATPase and associated intermediate reactions. Membranes incubated with gamma-[32P]ATP show the rapid incorporation of 32P into phosphoprotein. Phosphoprotein levels were markedly reduced (1) when ATP hydrolysis went to completion or (2) upon addition of unlabeled ATP, thus suggesting the participation of a rapid turnover phosphorylated intermediate in the gastric microsomal ATPase. Addition of K+, Rb+ or Tl+ greatly reduced the level of the intermediate while stimulating ATPase activity; the observed affinities of these cations were similar for the effects on both ATPase and intermediate levels, with Tl+ greater than K+ greater than Rb+. Neither ATPase nor intermediate were stimulated by Na+, and ouabain was without effect on the reactions, thus differentiating this system from the (Na+ + K+)-ATPase. Addition of various inhibitors showed differential effects on the partial reactions of the gastric ATPase system. N-ethylmaleimide and Zn2+ showed characteristics of completely abolishing the K+-stimulated component of ATPase as well as the effects of K+ in reducing the level of intermediate, thus suggesting that these agents exert their inhibitory effect on a phosphoprotein phosphatase partial reaction. F- abolished the K+-stimulated ATPase, but its more complex effects on the intermediate suggested an additional reaction step within the domain of the phosphorylated intermediate. Results are consistent with a model system for the gastric microsomal ATPase involving a Mg2+-dependent protein kinase, a phosphorylated intermediate(s), and a K+-stimulated phosphoprotein phosphatase.
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PMID:Studies on the phosphorylated intermediates of a K+-stimulated ATPase from rabbit gastric mucosa. 0 43

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.
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PMID:Some properties of purified phosphoprotein phosphatases from rabbit liver. 20 Feb 72

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.
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PMID:The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000. 21 Nov 35

Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K+; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkeable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.
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PMID:Phosphoprotein phosphatase activity at the outer surface of intact normal and transformed 3T3 fibroblasts. 22 67

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.
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PMID:Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex. 23 Sep 63

Callus calcifying cartilage alkaline phosphatase was resolved by DEAE-cellulose column chromatography into two distinct phsophatase activities. The phosphatase activity which was eluted first from the column, (phosphatase I), was active towards a variety of phosphate esters, sodium pyrophosphatase and several linear polyphosphates, while the second phosphatase activity , (phosphatase II), was active toward simple phosphate esters but not towards sodium pyrophosphate and linear oligo or polyphosphates. All the phosphate esters, sodium pyrophosphate and polyphosphates at higher concentrations were inhibitory for phosphatase I. The modulating effects of magnesium, calcium, zinc and other phosphatase modulators have been investigated. Both phosphatases from callus calcifying cartilage were found to be substrates of neuraminidase with sialic acid as the product. Besides the difference in their specificity, the phosphatases were found to be immunologically different and to have different molecular weights, strong indication that they are different enzymes.
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PMID:Resolution, purification and characterization of the orthophosphate releasing activities from fracture callus calcifying cartilage. 23 99

Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2-mercaptoethanol stimulated the enzyme activity, whereas N-ethylmaleimide and N-phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nuclsoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15--20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic-AMP-dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate-solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic-AMPstimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).
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PMID:Solubilization and characterization of phosphoprotein phosphatase(s) from bovine corpus-luteum plasma membranes. 24 Jun 98

The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.
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PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells. 131

A phosphotyrosyl protein phosphatase (PTPase) activity has been characterized in the plasma membranes of confluent AR42J pancreatic tumor cells using 32P-labeled poly(Glu, Tyr) as substrate. Membrane PTPase activity exhibited an apparent Michaelis constant of 3 microM and an apparent maximal velocity of 0.9 nmol.min-1.mg-1. It was inhibited by orthovanadate, zinc, poly(Glu,Tyr) and was stimulated by EDTA and dithiothreitol. Gel filtration of solubilized plasma membranes gave a peak of enzyme activity at a relative molecular weight of 70,000. Plasma membrane PTPase activity was changed during AR42J cell growth. At the beginning of culture, the control PTPase activity was minimal. Over the 5 days of culture, PTPase activity increased to reach a maximum (3.5-fold over control activity) preceding confluency by 2 days. Then the high level of PTPase activity was sustained until confluency. Incubation of the cells with the stable somatostatin analogue SMS 201-995 (SMS) resulted in a rapid and transient activation of crude membrane PTPase activity. Activation reached a maximum level within 5 min of addition and return to control levels within 20 min. The effect of SMS was dose dependent with half-maximal and maximal activation occurring at 6 pM and 0.1 nM SMS respectively.
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PMID:Characterization of a membrane tyrosine phosphatase in AR42J cells: regulation by somatostatin. 135 86

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.
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PMID:Effect of zinc on calmodulin-stimulated protein kinase II and protein phosphorylation in rat cerebral cortex. 164 55


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