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)

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.
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PMID:Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards. 132 16

A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activity has been identified and purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass was 20,000 Da. The protein lost its inhibitory properties when incubated with trypsin or treated with ethanol. The inhibitor protein does not inhibit type 1 phosphatase when either phosphorylase or hydroxymethylglutaryl-CoA reductase is the substrate. In contrast, this protein inhibitor inhibits the rat liver type 2A phosphatase activity when hydroxymethylglutaryl-CoA reductase is the substrate but not when phosphorylase a is the substrate. The inhibitor protein is not activated by incubation with ATP and cyclic AMP-dependent protein kinase and it is not phosphorylated by glycogen synthase kinase-3. These results, together with those of the kinetic experiments, suggest that the reductase phosphatase inhibitor is distinct from protein phosphatase inhibitor-1 and inhibitor-2.
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PMID:Purification and properties of a protein inhibitor that inhibits phosphatase 2A activity when hydroxymethylglutaryl coenzyme A reductase is the substrate. 254 29

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

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.
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PMID:Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase. 300 73

The synthetic phosphohexapeptides Arg-Arg-Ala-Thr(35P)-Val-Ala and Arg-Arg-Ala-Ser(32P)-Val-Ala, phosphorylated by the cAMP-dependent protein kinase and differing only in the nature of the phosphorylated residue, have been used as substrates of a partially purified rat liver protein phosphatase-T, distinct from the multifunctional protein phosphatase-1. While the phosphothreonyl hexapeptide is readily dephosphorylated (exhibiting a Km = 15 microM), the phosphoseryl one is almost unaffected. Such a behavior is not shared by protein phosphatase-1, calf intestine alkaline phosphatase, and potato acid phosphatase, all of which are more active on the phosphoseryl hexapeptide. The NH2-terminal basic residues critical for cAMP-dependent phosphorylation are not required in the dephosphorylation reaction, as both Arg can be removed without impairing the efficiency of protein phosphatase-T toward the phosphothreonyl peptide. On the other hand, the replacement of 2 Pro for the Ala and Val flanking Thr(32P), to give a new phosphohexapeptide reproducing the phosphorylated site of protein phosphatase inhibitor-1, prevents the protein phosphatase-T activity. Moreover, IgG heavy chain 32P labeled in tyrosine is not affected by protein phosphatase-T, while it is dephosphorylated by alkaline phosphatase. These results would indicate that protein phosphatase(s)-T represent a distinct class of protein phosphatases specifically involved in the dephosphorylation of phosphothreonyl residues fulfilling definite structural requirements.
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PMID:Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase. 628 35

Reticulocyte lysates contain two major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include (i) chromatographic elution profiles on DEAE-cellulose; (ii) specificity of lysate phosphatases toward [(32)P]phosphorylase a and [(32)P]phosphorylase kinase; (iii) sensitivity of lysate phosphatases to Mg(2+) ATP; and (iv) sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (a) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (b) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (c) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the alpha-subunit (38,000 daltons) of eIF-2 (eIF-2alpha) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (d) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (e) Inhibitor-2 has no effect on the activation in vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2alpha kinases. We propose that the inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type 1 eIF-2alpha phosphatase activity, which permits a basal eIF-2alpha kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2alpha and an inhibition of protein synthesis.
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PMID:Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates. 629 14

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].
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PMID:The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. 630 28

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.
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PMID:Purification of a latent protein phosphatase from rabbit skeletal muscle. 632 69

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the RII subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.
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PMID:A capillary electrophoresis-based assay for protein kinases and protein phosphatases using peptide substrates. 797 76

The unicellular marine dinoflagellate, Prorocentrum lima, an established producer of okadaic acid (OA), was shown to contain a type-1 protein phosphatase (PP-1) the biochemical profile of which on Mono-Q and Superdex-75 fast protein liquid chromatography was identical to the catalytic subunit of PP-1 from rabbit skeletal muscle. Purified P. lima PP-1 (apparent molecular mass 37.5 kDa) was highly sensitive to inhibition by mammalian protein phosphatase inhibitor-1 and inhibitor-2, and to OA itself. A 6-7-fold increase in OA production by P. lima, when grown under controlled conditions, correlated with an up to 300-fold increase in P. lima PP-1 activity. Furthermore, P. lima did not contain any detectable type-2A protein phosphatase activity. This study represents the first identification of a serine/threonine protein phosphatase in a dinoflagellate.
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PMID:Identification and characterisation of a type-1 protein phosphatase from the okadaic acid-producing marine dinoflagellate Prorocentrum lima. 822 16


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