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

Evidence is presented on the existence of an acid phosphoprotein phosphatase (APPase) associated with rat splenic cell nucleoli. The enzyme is purified 1250-fold from 0.3 M NaCl nucleolar extract by means of chromatography on P cellulose and Sephacryl S-200. The nucleolar acid phosphoprotein phosphatase is a very basic protein (pI 8.3) and shows maximal activity at pH 5.8. It dephosphorylates acidic phosphoproteins (casein and phosvitin), ATP, and p-nitrophenyl phosphate, but not basic phosphoproteins (histones and protamine phosphate). The enzyme activity is very dependent on reducing agents, especially on ascorbic acid. Divalent and monovalent cations did not affect phosphatase activity, but heavier divalent metals, Co2+ and Zn2+, strongly inhibit the enzyme activity. The activity was also inhibited by N-ethylmaleimide, indicating a requirement for free sulfhydryl groups. The estimated molecular weight of the purified enzyme is approximately 38,000 by gel filtration and sedimentation in sucrose gradient concentration.
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PMID:Characterization of low-molecular-weight acid phosphoprotein phosphatase associated with rat splenic cell nucleoli. 609 79

Two nuclear phosphoprotein phosphatases (PPases I and II) that cause dephosphorylation of [32P]histone, have been partially purified from goat testis. The enzymic activity is associated with nucleoplasm and chromatin. PPase I is markedly stimulated (approx. 200-600%) by Mg2+ or Mn2+ (1 mM) whereas Ca2+ (1 mM) causes slight stimulation (approx. 35%) of the enzyme. On the contrary, PPase II is only slightly activated (20-40%) by these metal ions (5 mM). Both the phosphoprotein phosphatase isoenzymes are maximally active at pH 6-7. PPases I and II are strongly inhibited (approx. 60-100%) by ZnCl2 (1 mM), P1 (5 mM) and thiol reagents. NaF (5 mM) inhibits (approx. 40%) specifically the activity of PPase I rather than PPase II. PPases are strongly inhibited by relatively high concentration of NaCl (0.4 M), isoenzyme II being more sensitive (approx. 80%) than isoenzyme I (approx. 50%). In addition to histones, both the isoenzymes can as well cause dephosphorylation of protamine, casein, and testicular nuclear proteins. Enzymic characteristics of the testicular nuclear PPases are clearly different from those of the cytosolic enzyme previously characterized.
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PMID:Characterization of nuclear phosphoprotein phosphatases from goat testis. 609 85

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60v-src. pp60v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl2, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60v-src activity by a vanadium-sensitive protein phosphatase activity.
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PMID:Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells. 609 87

Preincubation of two homogeneous rabbit liver phosphoprotein phosphatases (phosphophoprotein phosphohydrolases, EC 3.1.3.16) (Khandelwal, R.L., Vandenheede, J.R. and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) with ATP, ADP and PPi caused a time- and concentration-dependent inactivation of the enzyme activity. A 50% inactivation of phosphoprotein phosphatase I required relatively low concentration of inactivating metabolite and less preincubation time as compared to the inactivation of phosphoprotein phosphatase II. AMP, adenosine, adenine, Pi, EDTA, EGTA, 1,10-phenanthroline and diethyl dithiocarbamate were without effect on both enzymes. Pretreatment of both enzymes by metal-chelating agents followed by PPi did not augment the effect observed with PPi alone. Both inactivated enzymes could be reactivated by cobalt or manganese in the presence of dithiothreitol. Although the extent of reactivation by these two metal ions was almost similar, cobalt required a ten times lower concentration than manganese for this process. No difference in inactivation or reactivation of both enzymes was observed with different substrates, phosphorylase a, histone or casein, employed in the assay. Pi and PPi added during the assay inhibited activities of both phosphatases with phosphorylase a and casein substrates. With histone as substrate, PPi slightly inhibited enzyme activities at lower concentrations (0.01-0.25 mM) but activated at higher concentrations. Pi activated both enzymes with this substrate; maximal activation being observed at a concentration of 5 mM.
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PMID:Studies on in activation and reactivation of homogeneous rabbit liver phosphoprotein phosphatases by inorganic pyorphosphate and divalent cations. 624 57

A low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been purified to homogeneity from rabbit heart by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34,000. The S20,w value and Stockes radius for the enzyme were 3.45 and 24.0 A, respectively. Using these two values, a molecular weight of 35,000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. A heat-stable protein inhibitor for this enzyme with phosphorylase a as the substrate was also shown to be present in crude extracts of rabbit heart. It inhibited the dephosphorylation of phosphorylase a by phosphoprotein phosphatase by decreasing the Vmax of the reaction.
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PMID:Purification and properties of phosphoprotein phosphatase from rabbit heart. 624 38

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.
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PMID:Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes. 625 May 98

The Ca2+-dependent regulator protein (CDR), also frequently termed "calmodulin" was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1, 2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increase the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities of CDR. Dephosphorylation of casein and phosphorylase alpha by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1, 2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 microM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent Km of the phosphatase for the various histones 2- to 6-fold, while maximal velocities were 4- to 12-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by chelation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(L-aspartate) or poly(L-glutamate). The phosphorylated fragment from the NH2-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of dephosphorylation being reduced 3- to 6-fold by Ca2+ in incubations containing CDR.
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PMID:Interaction of calmodulin with histones. Alteration of histone dephosphorylation. 625 89

A protein phosphatase was isolated from the yeast, Candida utilis, which could reactivate (dephosphorylate) the phosphorylated form of the NAD-dependent glutamate dehydrogenase. The protein could also dephosphorylate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase). Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and L-glutamate; 2-oxoglutarate, NH+4 and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride.
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PMID:Reactivation of the phospho form of the NAD-dependent glutamate dehydrogenase by a yeast protein phosphatase. 626 12

Phosphoprotein phosphatases (phosphoprotein phosphohydrolase, EC 3.1.3.16) were partially purified from bovine thyroid with phosphorylated mixed histones, H1 histone and casein as substrates. Utilizing DEAE-cellulose chromatography, (NH4)2SO4 precipitation, gel filtration before and after freeze-thawing in 0.2 M 2-mercaptoethanol and histone-Sepharose chromatography, four fractions of enzyme activity were obtained and were designated as phosphatases I, IIA, IIB, and III. Phosphatases I had an apparent molecular weight of 155,000 and was dependent on Mn2+ for maximal activity. The enzyme had the greatest activity with histone H1 and was greatly stimulated by NaCl with phosphohistones as substrate. Phosphatases IIA and IIB had a molecular weight of about 70,000, were stimulated over 5-fold by Mn2+ and had much higher activities with phosphohistones than with casein in the presence of the cation. Phosphatase III, a possible catalytic subunit of larger molecular weight forms, had an apparent molecular weight of 30,000, was generally independent of Mn2+ and had high activities using all three substrates. Phosphatases I, IIA, and III were inhibited in a dose-dependent manner by sodium pyrophosphate (PPi), ATP, potassium phosphate (Pi) and sodium fluoride (NaF) when they were added directly to the reaction mixture with phosphorylated mixed histones as substrate. PPi was the most potent inhibitor and phosphatase III was the most sensitive to inhibition. PPi, ATP and NaF probably inactivated phosphatase III activity by removing an essential metal ion. After extensive dialysis to remove these inhibitors, the inactivated enzyme could be fully activated by Mn2+, but not by Mg2+, Ba2+, Cu2+, Cd2+, Ca2+, Zn2+ and Fe2+. Whereas the enzyme pretreated with Pi retained about 80% activity after dialysis, its activity was not further stimulated by Mn2+. The inactivated (demetallized) enzyme was less reactivated by Mn2+ in the presence of mM concentration of Pi. Moreover, the Mn2+-reactivated enzyme was again inactivated by Pi, NaF and ATP. Among them Pi was the most potent inactivator. These results suggest that Pi may have another inhibitory effect on metal ion binding besides on substrate binding and also that phosphatase III might be a metalloenzyme. In bovine thyroid, there are at least two major phosphoprotein phosphatases which may have different properties. Metal ion stimulation of phosphatase I and IIA activities may be through an interaction with the substrate or with a metal ion binding site on the regulatory subunit. The lowest molecular weight enzyme (phosphatase III) probably does not exist naturally in the cell.
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PMID:Discrimination of multiple forms of phosphoprotein phosphatase in bovine thyroid. 629 68


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