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)

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.
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PMID:Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis. 21 41

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

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.
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PMID:Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B. 299

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

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.
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PMID:Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei. 301 41

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.
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PMID:Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria. 303 Oct 42

A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.
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PMID:Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria. 303 Oct 43

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca2+ or Mn2+. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn2+; Co2+ and Ni2+ partially substituted for Mn2+, but Ca2+, Mg2+ and Zn2+ were ineffective. The stimulating effect of Mn2+ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn2+ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn2+) enzyme.
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PMID:Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity. 608 83

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.
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PMID:Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase. 627 90

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.
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PMID:Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase. 629 3


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