Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

An acid phosphatase has been partially purified from lactating bovine mammary gland. Properties of this enzyme were compared with those of a well-characterized phosphoprotein phosphatase from bovine spleen. The two enzymes were similar in their activation by sulfhydryl reagents and inhibition by metal chelating agents. Both enzymes rapidly hydrolyze ATP and aromatic phosphates and are relatively inactive toward alkyl phosphates; both are tartrate-resistant phosphatases. The mammary enzyme has a low Michaelis constant for alpha s1-casein (42 microM), and thus, like the spleen enzyme, appears to be a phosphoprotein phosphatase. Finally, the spleen and mammary enzymes displayed reactivity toward phosphotyrosine, a model substrate for phosphotyrosyl protein phosphatase. Thus, the phosphatases from spleen and mammary gland are quite similar in reactivity and could possibly be similar in function.
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PMID:Purification and properties of an acid phosphoprotein phosphatase from lactating bovine mammary gland with activity toward phosphotyrosine. 245 49

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.
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PMID:Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity. 246 73

Ca2+-calmodulin-dependent protein phosphatase activity is found in cytoskeletons of Y-1 mouse adrenal and bovine fasciculata cells. The activity is inhibited by three inhibitors of calmodulin (trifluoperazine, W-7 and pimozide) with EC50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn2+ and pyrophosphate, stimulated by Mn2+ and found to be tightly bound to the cytoskeleton. Substrates for endogenous phosphatase activity were defined by one- and two-dimensional polyacrylamide gels. Phosphatase activity was seen with proteins that are substrates for both cyclic AMP-dependent and cyclic AMP-independent kinase enzymes. One specific Ca2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-1 cells. The enzyme was found to be a heterodimer (MW 61,000 and 16,000) and the smaller subunit was shown to cross-react with antibodies raised against calcineurin from bovine brain. The purified enzyme catalyzes dephosphorylation of proteins (phosphorylase kinase and casein), phosphoamino acids (tyr greater than thre greater than ser) and a synthetic substrate (p-nitrophenyl phosphate). In addition, a new application of membrane transfer was devised by which the purified enzyme was incubated with a Western blot of cytoskeleton following incubation with [32P]ATP. This method defined four specific substrates of the enzyme (MW 150,000, 55,000, 35,000 and 30,000). Anti-calcineurin revealed that only a single Ca2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca2+-calmodulin-dependent phosphatase activity. 254 40

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.
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PMID:Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221. 254 89

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.
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PMID:Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II. 255 37

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCSH (390 kDa), PCSM (250 kDa) and PCSL (200 kDa) phosphatases. The PCSH phosphatase could be resolved into a 3(65:55 35 kDa)-subunit PCSH1 phosphatase and a 2(65:35 kDa)-subunit PCSH2 enzyme probably derived from the PCSH1 phosphatase, both characterized as specific deinhibitor phosphatases. PCSM phosphatase, a 3(72:65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCSL phosphatase is characterized by a 2(65:35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCSM greater than PCSH1 greater than PCSH2 greater than PCSL greater than PCSC) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (alpha-subunit) are choice substrates for these enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The polycation-stimulated protein phosphatases: regulation and specificity. 282 47

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

Previous reports have shown that heparin is an inhibitor of casein kinase-2 (CK-2). It is unclear whether heparin is also an inhibitor of glycogen synthase (casein) kinase-1 (CK-1), a type 1 casein kinase. In this study it is shown that CK-1 is potently inhibited by heparin when phosvitin or calcineurin are used as substrates. With casein as a substrate, however, the kinase is insensitive to inhibition by heparin. Using phosvitin as a substrate half-maximal inhibition of CK-1 was observed with 0.14 microgram/ml heparin. Kinetic analyses indicate that at a constant concentration (0.10 mM) of ATP the Km of CK-1 for phosvitin is increased eightfold in the presence of 0.9 microgram/ml heparin; the Vmax is unchanged with or without heparin. At a constant concentration of phosvitin (4 mg/ml) heparin (0.9 microgram/ml) decreased the Vmax for ATP by 57%; the Km is unchanged with or without heparin. The inhibition of CK-1 by heparin can be reversed by KCl (greater than 100 mM). These results indicate that heparin is a potent inhibitor not only of CK-2 but also of CK-1. Hence heparin inhibition can no longer be arbitrarily used as a criterion to discriminate between these kinases.
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PMID:Inhibition of glycogen synthase (casein) kinase-1 by heparin. 282 38

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, beta-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.
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PMID:Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa. 282 47

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.
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PMID:Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A. 282 78


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