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 phosphoprotein phosphatase that dephosphorylates smooth muscle myosin has been purified to apparent homogeneity from turkey gizzards. Smooth muscle phosphatase (SMP) IV has a molecular weight of 150,000 as determined by gel filtration on a Sephadex G-200 column and is composed of two subunits (Mr = 58,000 and 40,000). Although it is active toward a number of proteins, its activities toward the contractile proteins, intact myosin, heavy meromyosin, and isolated myosin light chains are higher than its activities toward phosphorylase alpha, histone IIA, and phosphorylase kinase. SMP-IV preferentially dephosphorylates the beta-subunit of phosphorylase kinase. The properties of the enzyme have been studied using heavy meromyosin, a soluble chymotryptic fragment of myosin, and isolated myosin light chains as substrates. SMP-IV has high affinity for both substrates and is optimally active at neutral pH. Divalent cations, Ca2+ and Mg2+, activate the dephosphorylation of heavy meromyosin but inhibit the activity toward myosin light chains. Low concentrations of ATP (1-5 mM) activate SMP-IV but concentrations higher than 5 mM are inhibitory. Inhibition of 50% of the activity of the enzyme by NaF and PPi requires concentrations higher than 10 mM. Rabbit skeletal muscle heat stable inhibitor-2 has no effect on the activity of SMP-IV toward heavy meromyosin, myosin light chains, and phosphorylase alpha.
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PMID:Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. 299 73

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.
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PMID:Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver. 299 4

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.
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PMID:Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas. 299 47

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.
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PMID:The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1. 300 40

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

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.
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PMID:Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations. 301 36

Rat liver nuclei contain a protein phosphatase that is indistinguishable from the catalytic subunit of protein phosphatase-1 in its molecular mass, sensitivity to inhibitor-1 and inhibitor-2 and specificity for the beta-subunit of phosphorylase kinase. This activity is not bound to the outer nuclear membrane, but located within the nucleus. The average level of protein phosphatase-1 activity in nuclei is at least 5-fold higher than its average extranuclear concentration.
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PMID:Identification of high levels of protein phosphatase-1 in rat liver nuclei. 301 74

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

Leishmania donovani promastigotes contain intense tartrate-resistant cell surface acid phosphatase (ACP1) which blocks superoxide anion production by activated human neutrophils [A.T. Remaley et al. (1984) J. Biol. Chem, 259, 11173-11175]. An extensively purified preparation of ACP1 dephosphorylates several phosphoproteins which are phosphorylated at serine residues; these include: pyruvate kinase (Km 1.6 microM; Vmax 71.4 U (mg protein)-1), phosphorylase kinase (Km 0.076 microM; Vmax 5.4 U (mg protein)-1) and histones (Km 4.86 microM; Vmax 2.2 U (mg protein)-1). However, the specific activity of the leishmanial phosphatase on these phosphoproteins is very low as compared to other phosphoprotein phosphatases. The phosphatase activity of ACP1 was also low on phosphohistone phosphorylated at tyrosine residues. Phosphatidylinositol-4,5-diphosphate (PIP2) and inositoltriphosphate (IP3) were also tested as ACP1 substrates. PIP2 was hydrolyzed rapidly by ACP1. The rate of hydrolysis of PIP2 was higher at pH 6.8 (Km 2.35 microM; Vmax 107 X 10(3) U (mg protein)-1) than at pH 5.5 (Km 4.16 microM; Vmax 71 X 10(3) U (mg protein)-1). 32P-labeled IP3 was also a substrate for ACP1; the hydrolysis products consisted of a mixture of inositoldiphosphate and inositolmonophosphate. ACP1 and ten other phosphatases were tested for their ability to dephosphorylate proteins and to inhibit O2- production by stimulated human neutrophils. There was no correlation between the protein phosphatase activity of the acid- and alkaline phosphatases and their ability to block neutrophil O2- production. The results indicate that ACP1 probably blocks the production of reduced oxygen intermediates by a mechanism that does not involve dephosphorylation of phosphoproteins; however, the possibility that the parasite's phosphatase affects phagocyte metabolism by degrading PIP2 or IP3 should be considered.
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PMID:Hydrolysis of phosphoproteins and inositol phosphates by cell surface phosphatase of Leishmania donovani. 301 59

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.
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PMID:Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody. 302 48


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