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 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 type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.
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PMID:Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. 283 Dec 1

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases-2A0, 2A1 and 2A2 by anion-exchange chromatography, gel-filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose-1,6-bisphosphatase, pyruvate kinase, acetyl-CoA carboxylase and phenylalanine hydroxylase by phosphatases-2A1 and 2A2, and with several of these substrates activation was even more striking (20-100-fold) than that observed with phosphorylase (approximately 5-fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit. The dephosphorylation of phosphorylase by protein phosphatase-1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl-CoA carboxylase were markedly stimulated by histone H1 and protamine (2-13-fold). Consequently, with the appropriate substrates, protein phosphatase-1 can also be regarded as a basic-polypeptide-activated protein phosphatase. Heparin stimulated (1.5-2-fold) the dephosphorylation of phosphorylase by phosphatases-2A0 and 2A1, provided that Mn2+ was present, but phosphatase-2A2 and the free catalytic subunit of phosphatase-2A were unaffected. Heparin, in conjunction with Mn2+, also stimulated (1.5-fold) the dephosphorylation of glycogen synthase (labelled in sites 3 abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase-2A1, but not by phosphatase-2A2. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase-1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase-1 was stimulated by this mucopolysaccharide. The studies demonstrate that basic proteins can be used to distinguish protein phosphatase-1 from protein phosphatase-2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 micrograms/ml protamine or at heparin concentrations greater than 150 microM.
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PMID:The protein phosphatases involved in cellular regulation. 1. Modulation of protein phosphatases-1 and 2A by histone H1, protamine, polylysine and heparin. 298 84

Protein phosphatases involved in cellular regulation have been categorized functionally into two major types by substrate specificity and sensitivity to protein inhibitors. In this classification type-1 phosphatases are inhibited by the heat-stable protein inhibitor-2 (I-2), whereas type-2 phosphatases are considered insensitive to inhibition by this protein. This study demonstrates that the phosphorylase phosphatase activity of both purified type-1 and type-2 catalytic subunits can be blocked by micromolar concentrations of I-2. Heparin also was more effective at inhibiting the type-1 compared to type-2 phosphatase but required thousandfold higher concentrations than I-2. The specificity of the interaction with I-2 indicates that the tertiary structures of the two phosphatase catalytic subunits closely resemble one another. However, only the type-1, not the type-2, protein phosphatase activity was neutralized by immunoglobulins affinity-purified against the Mr = 33,000 catalytic fragment of the type-1 phosphatase. Preparations of rabbit skeletal muscle type-1 phosphatase catalytic fragment and of bovine cardiac type-2 phosphatase catalytic subunit were compared by "Western" immunoblotting with sheep polyclonal and mouse monoclonal immunoglobulins raised against the respective proteins. Monoclonal anti-type-2 immunoglobulins preferentially stained the type-2 phosphatase catalytic subunit used as antigen, but displayed cross-reaction with 10-50 times more of the type-1 phosphatase. In contrast, as found with their effects on activity, sheep anti-type-1 immunoglobulins were specific; immunoblotting detected the type-1, not the type-2, catalytic protein. We conclude that the two catalytic proteins have at least one common primary structural epitope recognized by the monoclonal immunoglobulins. These data, taken together with other recent immunochemical results, support a hypothesis that this family of enzymes was derived from a common ancestral protein phosphatase catalytic subunit.
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PMID:Protein phosphatase type-1 and type-2 catalytic subunits both bind inhibitor-2 and monoclonal immunoglobulins. 302 55

Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase-1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin-Sepharose CL-6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor-1. The phosphatase bound to heparin-Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor-1.
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PMID:Heparin inhibits the activity of protein phosphatase-1. 632 37

Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.
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PMID:Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney. 658 97

Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory subunits (B). Immunological analyses identified B alpha/PR55 alpha as the major regulatory subunit of brain PP2A while a unique B' subunit was associated with the cardiac enzyme. Recombinant PP2A heterotrimers were purified from insect cells infected with baculoviruses expressing A and C, in combination with viruses expressing B alpha/PR55 alpha, B beta/PR55 beta, or SV40 small tumor antigen (st). Phosphatase activities of rAC-B alpha and rAC-B beta were similar to those for brain AC-B alpha, while rAC-st was 50-80% less active. Heparin had no effect on rAC-st myosin light chain phosphatase activity, while the B subunit-containing forms were stimulated 2-3-fold. Protamine caused a 3-4-fold increase in AC-B alpha and rAC-st activities and a marked activation of rAC-B beta (6-fold) and AC-B' (10.5-fold). When histone H1 was used as substrate, all of the heterotrimers were stimulated approximately 4-fold by heparin. The activity of AC-B' and rAC-B beta were increased 2-fold by Mn2+, while a 6-fold stimulation was observed with rAC-st. Chemical cross-linking of AC-B alpha and AC-B beta generated 200-kDa complexes, while AC-st was present as a 150-kDa complex. These results demonstrate that different regulatory proteins affect enzyme activity and the response to agents that modify PP2A activity in vitro. Different PP2A heterotrimers are likely to have distinct functions in vivo, and changes in subunit composition will have an important impact on signal transduction pathways.
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PMID:Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. 805 Nov 2

Protein phosphatase 2A2 is inactivated by phosphorylation following incubation with purified preparations of an autophosphorylation-activated protein kinase (Hong Guo and Zahi Damuni (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504). This protein kinase was purified about 250,000-fold from extracts of bovine kidney to apparent homogeneity. The purified preparations exhibited a single polypeptide of apparent M(r) approximately 36,000. Up to 1 mol of phosphoryl groups was incorporated per mol of the purified kinase following incubation with ATP. This autophosphorylation reaction (t1/2 approximately 0.5-1 min) was accompanied by a approximately 10-fold activation of the kinase. Autophosphorylation and activation were reversed by protein phosphatase 2A2 or the catalytic subunit of protein phosphatase 1. Phosphoamino acid analysis indicated that the kinase underwent autophosphorylation on threonines. The rate of autophosphorylation was independent of the concentration of the enzyme and a slope of 0.97 (gamma = 0.998) was obtained by van't Hoff's plot indicating that autoposphorylation was intramolecular. Relative to myelin basic protein, the enzyme exhibited about 8, 62, 130, 33, 5, and < 0.1% activity with histones H1, H2A, H2B, H3, and H4 and with glycogen synthase alpha, respectively. Heparin inhibited the activity of the enzyme half-maximally at about 20 micrograms/ml. The results indicate that this autophosphorylation-activated kinase is a new protein kinase.
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PMID:Purification and characterization of an autophosphorylation-activated protein serine threonine kinase that phosphorylates and inactivates protein phosphatase 2A. 838 87

The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
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PMID:Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity. 865 37