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)

Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.
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PMID:Nuclear phosphoprotein phosphatase from calf liver. 3 41

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.
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PMID:Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP. 16 13

A protein whose endogenous phosphorylation and dephosphorylation are affected by cAMP has been found in the soluble and particulate fractions of all vertebrate tissues studied. This phosphoprotein, which contained a substantial proportion of the radioactive phosphate observed on SDS-polyacrylamide gels, was estimated to have an apparent molecular weight of 49,000. In the presence of Zn++, cAMP inhibited the endogenous phosphorylation of this protein (protein 49) in the cytosol and microsomal fractions. In the presence of Mg++, cAMP stimulated the phosphorylation of protein 49 in the cytosol fractions, but had only slight effects in the microsomal fractions. The dephosphorylation of protein 49 by an endogenous protein phosphatase was markedly stimulated by cAMP in the cytosol and microsomal fractions of all tissues studied. The binding of 8-azido-cAMP (a photoaffinity analog of cAMP, which reacts specifically with cAMP-binding sites) to subcellular fractions was also studied. This binding was principally to a protein of molecular weight 49,000. These and other data suggest that a cAMP-binding protein with a molecular weight of 49,000 capable of undergoing cAMP-dependent phosphorylation and dephosphorylation, occurs in a variety of tissues.
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PMID:Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation. 16 55

Calcium transport by cardiac sarcoplasmic reticulum (SR) was compared in hyperthyroid (HT) and euthyroid (ET) rats. Both Ca2+ uptake (97 +/- 3.1 nmol/mg per min in HT vs. 63 +/- 2.9 nmol/mg per min in ET, P less than 0.01) and CA2+ -stimulated ATPase activity (61 +/- 4.1 vs. 37 +/- 1.6 nmol Pi/mg per min, P less than 0.01) were higher in the thyroxine-treated animals. These changes were accompanied by enhanced cyclic AMP-dependent phosphorylation of cardiac SR in hyperthyroid rats (180 +/- 4.3 pmol Pi/mg per min vs. 117 +/- 4.2 pmol Pi/mg per min, P less than 0.01). SDS-polyacrylamide gel electrophoresis of cardiac SR showed that phosphorylation of a 22,000-dalton protein (phospholamban) primarily accounted for the differences between the two groups. There was no difference in the rate of SR dephosphorylation by endogenous phosphoprotein phosphatase between HT and ET rats. Differences in cyclic AMP-dependent phosphorylation between the two groups were blunted in the presence of excess exogenous cyclic AMP-dependent protein kinase. These results suggest that increased levels or activity of endogenous cyclic AMP-dependent protein kinases may partially explain enhanced calcium transport by the cardiac SR of hyperthyroid animals.
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PMID:Enhanced phosphorylation of myocardial sarcoplasmic reticulum in experimental hyperthyroidism. 20 50

The delta-subspecies of protein kinase C (PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose Fast Flow, Phenyl 5PW, Heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was doublet with molecular weight of 78 kDa and 76 kDa on SDS-PAGE. This doublet proteins were separated partially by Mono Q column chromatography, both of which were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78 kDa protein was a phosphorylated form of the 76 kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid, and was purified for comparison. This recombinant enzyme was also doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mapping. In addition, these the enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, and Ca2+. Comparison was also made between the enzymological properties of delta PKC and alpha PKC, such as activation kinetics, sensitivity to protein kinase inhibitors and substrate specificity which were distinctly different from each other.
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PMID:Enzymatic properties of ubiquitously expressed delta-subspecies of protein kinase C differing from other members of protein kinase C family. 129 10

Two isoforms of calcineurin beta subunit(beta 1 and beta 2) were identified in rat testis by a monoclonal antibody Va1. Both beta 1 and beta 2 were recovered in calmodulin binding protein fraction and showed calcium shift on SDS-polyacrylamide gel electrophoresis which is the specific character for EF-hand calcium binding protein. beta 2 showed same apparent molecular weight on SDS-PAGE as that of brain calcineurin beta and was found in wide variety of tissues. beta 1 was shown to have six amino acid polypepeptide sequence and it showed higher molecular weight than brain beta and was specific for testis.
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PMID:Identification of testis specific calcineurin beta subunit isoform by a monoclonal antibody and detection of a specific six amino acid sequence. 131 15

A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previously showed that cis-diamminedichloroplatinum(II) (CDDP)-resistant cells, PC-9/CDDP, were cross-resistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesized that the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylation of whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 32P, we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.
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PMID:Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II). 131 Apr 90

Nuclei from bovine thymus contain a high level of partially latent protein phosphatase 1 (PP-1). More than 90% of this PP-1 is associated with the insoluble chromatin/matrix fraction and can be extracted with 0.3 M NaCl. The salt extract also contains three heat- and acid-stable inhibitory proteins of PP-1 that can be resolved on Mono Q. We have purified two of these nuclear inhibitors of PP-1 (NIPP-1a and NIPP-1b) until homogeneity. They are acidic proteins (pI = 4.4) with a molecular mass of 18 kDa (NIPP-1a) and 16 kDa (NIPP-1b) on SDS-PAGE. Judged from the larger molecular mass that was deduced from gel filtration (35 kDa), NIPP-1a and NIPP-1b appear to be asymmetric or dimeric proteins. The nuclear inhibitors totally inhibited the phosphorylase phosphatase activity of PP-1, but even at a 250-fold higher concentration they did not affect the activities of the other major serine/threonine protein phosphatases (PP-2A, PP-2B, and PP-2C). NIPP-1a and NIPP-1b inhibited the catalytic subunit of PP-1 with an extrapolated Ki of about 1 pM, which is some three orders of magnitude better than the cytoplasmic proteins inhibitor 1/DARPP-32 and modulator. The nuclear inhibitors were not inactivated by incubation with protein phosphatases that inactivate inhibitor 1 and DARPP-32. Unlike modulator, they were not able to convert the catalytic subunit of PP-1 into a MgATP-dependent form. Remarkably, the extent of inhibition of PP-1 by NIPP-1b depended on the nature of the substrate. The phosphorylase phosphatase and casein phosphatase activities of PP-1 were completely blocked by NIPP-1b, whereas the dephosphorylation of basic proteins was either not at all inhibited (histone IIA) or only partially (myelin basic protein). These data may indicate that the acidic NIPP-1b is inactivated through complexation by basic proteins. Indeed, nonphosphorylated histone IIA antagonized the inhibitory effect of NIPP-1b on the casein phosphatase activity of PP-1. Our data show that the nucleus contains specific and potent inhibitory proteins of PP-1 that differ from earlier described cytoplasmic inhibitors. We suggest that these novel proteins may control the activity of nuclear PP-1 on its natural substrate(s).
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PMID:The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei. 132 7

The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.
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PMID:The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody. 132 57

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.
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PMID:Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules. 133 16


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