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)

A simplified procedure for the purification of low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been described from rabbit heart. The enzyme was purified to homogeneity by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34 000. The S20, W value and Stokes radius for the enzyme was 3.35 and 24.0 A(1 A = 0.1 nm), respectively. Using these two values, a molecular weight of 35 000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. Kinetic studies revealed the lowest Km with glycogen synthase D and maximum Vmax for the reaction with phosphorylase a.
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PMID:The properties of purified low molecular weight phosphoprotein phosphatase from rabbit heart. 23 96

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.
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PMID:Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex. 24 3

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.
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PMID:Phosphorylase kinase from human polymorphonuclear leukocytes. 44 42

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.
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PMID:The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme. 130 82

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.
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PMID:Characterization of neuronal protein phosphatases in Aplysia californica. 131 Jul 28

Elevated dosage of the GAC1 gene from the yeast Saccharomyces cerevisiae causes hyperaccumulation of glycogen whereas a gene disruption of GAC1 results in reduced glycogen levels. Glycogen synthase is almost entirely in the active, glucose 6-phosphate-independent, form in cells with increased gene dosage of GAC1 whereas the enzyme is mostly in the inactive form in strains lacking GAC1. GAC1 encodes an 88 kDa protein that is similar to the regulatory subunit (RG1) of phosphoprotein phosphatase type 1 (PP-1) from skeletal muscle that targets PP-1 to glycogen particles. Taken together, these results suggest that GAC1 encodes a regulatory subunit of PP-1. As previously shown for glycogen phosphorylase (GPH1), GAC1 RNA accumulates concomitantly with the appearance of glycogen. A strain with a mutation in the regulatory subunit of the cAMP-dependent protein kinase (bcy1) fails to accumulate GPH1 and GAC1 RNA. These results point to coordinate regulation of enzymes involved in glycogen metabolism at the level of RNA accumulation and indicate that at least part of this control is exerted by the RAS-cAMP pathway.
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PMID:GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae. 131 Sep 38

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.
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PMID:Evidence for the regulation of exocytic transport by protein phosphorylation. 131 11

Three phosphatases active on phosphocasein (PhosphoCasein Phosphatases) termed PCP-I, PCP-II and PCP-III were isolated from maize seedlings by DEAE-cellulose chromatography and were shown to display a different specificity toward a variety of phosphorylated substrates including pNPP, phosphohistones, phosphorylase a and several phosphopeptides containing either phosphoserine or phosphothreonine. PCP-I and PCP-II bind to heparin-Sepharose, retain a remarkable pNPP activity, are uncapable to dephosphorylate phosphorylase a, and display striking activity toward the acidic phosphopeptide AS[32P]EEEEE. They also by far prefer phosphoseryl peptide RRAS[32P]VA over its phosphothreonyl derivative and are unsensitive to okadaic acid up to 1 microM. These properties are not consistent with the belonging of PCP-I and -II to any of the known classes of protein phosphatases and suggest that they are acidic phosphatases. Conversely, PCP-III is essentially free of pNPP activity; it readily dephosphorylates phosphohistone H1 and phosphorylase a and it displays a striking preference toward the phosphothreonyl peptides (RRAT[32P]VA and RRREEET[32P]EEEAA), while the phosphoseryl peptides (RRAS[32P]VA and AS[32P]EEEEE) are very poor substrates of the enzyme. These properties together with the findings that PCP-III does not bind to heparin-Sepharose and is highly sensitive to okadaic acid (IC50 = 0.2 nM) allow to identify PCP-III with a protein phosphatase of the PP-2A class.
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PMID:Identification of protein phosphatase activities in maize seedlings. 131 1

p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl-terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation-stimulated (PCS/type-2A) protein phosphatase and the active catalytic subunit of the ATP,Mg-dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10-20-fold-better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100-fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72-kDa, 65-kDa and 55-kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8-fold) by the PCSH1 phosphatase and is about a 40-fold and 95-fold-better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl-terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km = 10 microM, Vmax = 3882 nmol.min-1.mg-1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor-1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.
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PMID:Specificity of the polycation-stimulated (type-2A) and ATP,Mg-dependent (type-1) protein phosphatases toward substrates phosphorylated by P34cdc2 kinase. 131 64

Proglycosyn, a phenylacyl imidazolium compound that lowers blood glucose levels, was demonstrated previously to promote hepatic glycogen synthesis, stabilize hepatic glycogen stores, activate glycogen synthase, inactivate glycogen phosphorylase, and inhibit glycolysis. In the present study proglycosyn was found to inhibit fatty acid synthesis, stimulate fatty acid oxidation, and lower fructose 2,6-bisphosphate levels, but to have no significant effects on cell swelling and the levels of cAMP in hepatocytes prepared from fed rats. Verapamil and atropine blocked the effects of proglycosyn on glycogen metabolism, but these compounds inhibit proglycosyn accumulation by hepatocytes. Proglycosyn stimulated phosphoprotein phosphatase activity in postmitochondrial extracts, as measured by dephosphorylation of phosphorylase a and glycogen synthase D, but this action required a very high concentration of the compound, making it unlikely to be the actual mechanism involved. It is proposed that a metabolite of proglycosyn is responsible for its metabolic effects.
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PMID:Metabolic effects of proglycosyn. 131 46


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