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)

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.
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PMID:Activation of brain calcineurin towards proteins containing Thr(P) and Ser(P) by Ca2+, calmodulin, Mg2+ and transition metal ions. 609 74

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.
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PMID:The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle. 609 84

The regional and cellular distribution of G-substrate, a 23,000-dalton protein substrate specific for guanosine 3',5'-cyclic monophosphate-dependent protein kinase, has been examined in mammalian brain using immunoprecipitation, radioimmunoassay, and peptide-mapping techniques. In rabbit brain, G-substrate was found to be highly concentrated in the cerebellum. The concentration of G-substrate in cerebellar cytosol was 27.2 pmol/mg. The concentrations of G-substrate in cortex, hippocampus, and caudate were only 1 to 2% of that found in cerebellum. Studies of neurological mutant mice lacking either Purkinje cells (PCD and nervous) or granule cells (weaver) suggested that, within the cerebellum, G-substrate is localized almost exclusively in Purkinje cells. A phosphoprotein present in noncerebellar brain regions, which co-migrated with G-substrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown by peptide mapping to consist predominantly of phosphatase inhibitor-1. Phosphatase inhibitor-1, a potent inhibitor of protein phosphatase-1, is known to share several physicochemical properties with G-substrate. In contrast to the results obtained with G-substrate, the concentration of phosphatase inhibitor-1 was significantly lower in cerebellum than in other major brain regions. These and other data suggest that G-substrate may be a Purkinje cell-specific protein phosphatase inhibitor.
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PMID:Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase. 609 45

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.
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PMID:The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein. 609 39

Amongst the several cyclic AMP-and Ca2+-independent synthase kinases that are reported by several laboratories, the kinase FA is unique in having a bifunctional nature: it can also promote the conversion of the inactive ATP,Mg-dependent protein phosphatase to its active form. By doing so, it produces an active multisubstrate phosphatase which reverses the major protein phosphorylations that control glycogen metabolism. In rabbit skeletal muscle, two forms of kinase FA can be distinguished, which seem to interconvert into one common bifunctional catalytic unit. It is proposed that the two molecular forms either reflect the existence of a regulatory subunit which dissociates during the purification, or suggest a reversible modification of the bifunctional catalytic unit. In order to serve a useful physiological role in the regulation of glycogen metabolism, the enzyme has to select between its two opposite activities in any given physiological condition, and the putative regulatory subunit or modification of the catalytic unit could play a fundamental role in this modulation. In vitro experiments have provided us with two proteins that are suitable candidates to discriminate between the two activities in FA: the heat stable phosphatase inhibitor-1 and the phosphatase modulator protein. Both can prevent the expression of the protein phosphatase activity; however, a well defined amount of modulator protein is absolutely required for an efficient activation of the FC-enzyme by FA, but an excess [M] decreases the rate as well as the extent of activation. This means that excess modulator protein can block the phosphatase activating capacity of FA. The same, or even higher, concentrations of modulator (or inhibitor-1) have absolutely no effect on the synthase kinase activity of the FA protein. No effector of this synthase kinase activity has yet been found, although by its shunting away from the phosphatase activation, more of the FA could be effective as a synthase kinase.
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PMID:Characterization of different forms of kinase FA from rabbit skeletal muscle. 610 May 85

Calcineurin, a calmodulin-binding protein from brain, has been shown to possess a metal ion-dependent and calmodulin-stimulated phosphatase activity towards phosphorylase kinase and inhibitor-1 (Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84). In this report, we show that calcineurin can also dephosphorylate p-nitrophenyl phosphate and free phosphotyrosine. However, calcineurin does not show significant activity towards phosphothreonine, phosphoserine, or several other low molecular weight phosphocompounds tested. As we have found with phosphorylase kinase and phosphocasein, the dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine is stimulated by calmodulin and is metal ion-dependent with the order of efficiency being Mn2+ much greater than Co2+ greater than Ca2+. The dephosphorylation of these substrates appears to be an intrinsic property of calcineurin and is not due to contamination by alkaline phosphatases since the pH optimum for calcineurin activity occurs at a neutral rather than an alkaline pH. The dephosphorylation of p-nitrophenyl phosphate provides an easy, rapid, and accurate method for the quantification of calcineurin activity as well as permitting insight into reaction kinetics. The dephosphorylation of free phosphotyrosine by calcineurin suggests that this compound may be a physiological substrate of calcineurin.
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PMID:Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin. 619 Aug 10

Protein phosphatase type 1 and type 2 activities (designated PP-1 and PP-2, respectively) from rabbit reticulocyte lysates have been identified and characterized based on criteria previously established for similar activities in rabbit skeletal muscle and rabbit liver. These include (a) chromatographic separation on DEAE-cellulose, (b) substrate specificity toward glycogen phosphorylase a and the alpha- and beta-subunits of phosphorylase kinase, (c) differential sensitivity to the heat-stable protein phosphatase inhibitors-1 and -2, and (d) sensitivity to MgATP. When total lysate phosphatases are assayed in the presence of 1 mM MnCl2, protein phosphatase type 2 represents 84% of lysate phosphorylase phosphatase activity. However, when phosphatase assays are carried out with MgATP concentrations similar to those in the lysate, type 2 activity is diminished, and the levels of type 1 (41%) and type 2 (59%) phosphatase activities are comparable. A small proportion (6%) of total lysate phosphatase is tightly bound to the ribosomes, where type 1 phosphatase predominates. At least five species of protein phosphatases can be identified in lysates. These constitute two forms of protein phosphatase type 1, one of which (designated FC) is dependent on MgATP and a lysate activator protein FA; both FC and FA have been identified previously in skeletal muscle. Three species of protein phosphatase type 2 have been identified and designated PP-2B, PP-2A1, and PP-2A2 based on criteria recently established for rabbit skeletal muscle and rabbit liver phosphatases, which display similar phosphatase profiles. Lysate protein phosphatases types 1, FC, 2A1, and 2A2 can all act on phosphorylase a and the alpha- (type 2) or beta-(type 1) subunit of phosphorylase kinase. PP-2B, a Ca2+/calmodulin-dependent phosphatase, specifically dephosphorylates the alpha-subunit of phosphorylase kinase, but does not act on phosphorylase alpha. The heat-stable protein phosphatase inhibitor-2 from skeletal muscle completely blocks the activity of the two type 1 phosphatases (PP-1, FC), but has no effect on the three species of type 2 protein phosphatase. A preliminary assay of the two heat-stable phosphatase inhibitors in lysates indicates significant levels of inhibitor-2, but little or no detectable inhibitor-1.
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PMID:Separation and identification of type 1 and type 2 protein phosphatases from rabbit reticulocyte lysates. 629 96

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.
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PMID:The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities. 630 24

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...
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PMID:The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism. 630 25


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