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 type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogen-protein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1C, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (I-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3. The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.
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PMID:The protein phosphatases involved in cellular regulation. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle. 298 73

In a previous communication (Waisman, D.M., Smallwood, J.I., Lafreniere, D. and Rasmussen, H. (1983) Biochem, Biophys. Res. Commun. 116, 435-441) we reported that chromatography of bovine brain 100,000 X g supernatant on diethylaminoethyl (DEAE) cellulose and analysis of resultant fractions by chelex competitive calcium binding assay, resolved three peaks of calcium binding activity. Gel permeation chromatographic analysis of each peak resolved apparent Mr 40,000 (Peak I), Mr 75,000, Mr 230,000 and Mr 420,000 (Peak II), and Mr 38,000 (Peak III). In the present communication the calcium binding proteins responsible for the calcium binding activity peaks resolved by gel permeation chromatography, have been purified and identified as caligulin, (Mr 40,000), calcineurin, (Mr 230,000) and calmodulin, (Mr 38,000). In addition, a novel calcium binding protein (Mr 48,000 by SDS PAGE) has been identified from the Mr 75,000 calcium binding activity peak.
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PMID:Identification of bovine brain Ca2+-binding proteins. 298 28

A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell.
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PMID:Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin. 298 99

The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.
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PMID:[Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties]. 299 59

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.
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PMID:Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex. 301 14

A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.
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PMID:Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit. 302 30

Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+. The phosphatase is strongly inhibited by heparin and fluoride. L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM. The molecular mass of the native phosphatase was found to be 180,000 Da. Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each. Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7. Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic [32P] phosphate was demonstrated.
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PMID:Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae. 302 61

Muscarinic receptor, from porcine synaptic membrane, was purified by affinity chromatography. Molecular weight analysis by SDS-gel electrophoresis revealed one major peptide with an apparent Mr of 68 +/- 2 Kda. The purified receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase resulting in a concomitant loss in specific binding, and this loss was reversed by calcineurin.
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PMID:Phosphorylation of brain muscarinic receptor: evidence of receptor regulation. 303 Mar 6

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.
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PMID:Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp. 303 Jul 44

A calmodulin-stimulated protein phosphatase has been purified from bovine myocardium. The purification procedure involves sequential DEAE-Sephacel ion exchange chromatography, calmodulin-Sepharose affinity chromatography, and high performance liquid chromatography using a Spherogel TSK DEAE 5PW column. By SDS polyacrylamide gel electrophoresis, the purified cardiac phosphatase consists of two subunits of Mr 61,000 and 19,000, similar to the brain enzyme, calcineurin. Protein phosphatase activity of the cardiac enzyme is stimulated by Ca2+-calmodulin and inhibited by the calmodulin antagonist drug, calmidazolium. Effects of a series of divalent cations on catalytic activity of the cardiac calmodulin-stimulated protein phosphatase are similar to those observed with calcineurin, when the two enzymes are assayed under identical conditions. Highly enriched preparations of bovine cardiac sarcolemma contain substrates of cAMP-dependent protein kinase of Mr 166 K, 133 K, 108 K, 79 K, 39 K, and 14 K, which are specifically dephosphorylated by the calmodulin-stimulated phosphatase with pseudofirst-order rate constants of 0.23, 0.46, 0.69, 0.35, 0.69, and 0.115 min-1, respectively. These substrates are not present in purified preparations of cardiac sarcoplasmic reticulum. These results support a role of the calmodulin-stimulated phosphatase in the Ca2+-regulation of specific sarcolemmal processes by protein dephosphorylation.
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PMID:Cardiac calmodulin-stimulated protein phosphatase: purification and identification of specific sarcolemmal substrates. 303 93


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