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)

We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phosphatase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p-hydroxymercuribenzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP-dependent protein kinase (cAMP-dPK) activity was present in both supernatant (S2) and particulate (P2) fractions, with the majority (60-85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP-dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory subunit of the cAMP-dPK, R-II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP-dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were detected, suggesting that the genetic defect in cAMP metabolism is not accompanied by constituent abnormalities in phosphorylated substrates in the adult fly, and that the physiological defects in these mutants result from aberrations in the interaction of the cAMP cascade with normal substrates. The majority of Ca2+/calmodulin kinase activity (80-90%, depending on the homogenization procedure) was associated with S2, as revealed by phosphorylation of exogenous synapsin I. Two endogenous substrates for this kinase in P2 had molecular masses of approximately 45 and 87 kilodaltons. At least 11 substrates for the Ca2+/calmodulin-dependent kinase were detected in S2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vitro protein phosphorylation in head preparations from normal and mutant Drosophila melanogaster. 304 Sep 7

Interactions of several divalent cations (Mn2+, Ca2+, Co2+, Sr2+, and Zn2+) with EGTA-inhibitable adenylate cyclase were investigated in washed membranes (particles) isolated from the gray matter of rat cerebral cortex. The EGTA-inhibitable (called sensitive) enzyme activity was assayed in the presence of Triton X-100 since this detergent caused a marked increase (up to 20-fold) in the enzyme activity. The effects of various divalent metals (all added as chloride salt) indicated the presence of two distinct sites called site I and site II. At low concentrations (less than micromolar) Mn2+, Co2+, and Ca2+ increased (up to 10-fold) the enzyme activity to the same extent and appeared to act via binding to site I (high affinity site). The rank order of affinity was Mn2+ greater than or equal to Co2+ greater than Ca2+. Zn2+ showed the highest affinity and Sr2+ the lowest towards binding to site I; both these metals increased the enzyme activity to lesser extents than Mn2+, Co2+, or Ca2+. GTP was not required for the stimulation of this enzyme by low concentrations of Ca2+. The interaction of Mn2+ with site II (low affinity site) caused further increase in the enzyme activity, whereas Co2+, Ca2+, and Sr2+ were inhibitory at concentrations greater than 10 microM. Isolated fraction contained loosely and tightly associated pools of calmodulin. Myelin basic protein, but not calcineurin, inhibited the EGTA-sensitive adenylate cyclase activity. The EGTA-insensitive enzyme activity was increased by norepinephrine by mechanisms that depended on GTP and was inhibited by Ca2+. The stimulation of the EGTA-insensitive enzyme modulated the Mg2+ requirement such that Mg2+ binding to the low affinity site (site II) apparently occurred with higher affinity. The likely significance of these results is discussed with regard to (i) the presence of two classes of adenylate cyclase in rat cerebral cortex gray matter and (ii) the regulation of their activities by calmodulin-requiring and GTP-requiring mechanisms.
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PMID:EGTA-sensitive and -insensitive forms of particulate adenylate cyclase in rat cerebral cortex: regulation by divalent cations and GTP. 393 3

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.
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PMID:Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+. 608 12

Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.
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PMID:Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose. 608 42

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca2+ or Mn2+. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn2+; Co2+ and Ni2+ partially substituted for Mn2+, but Ca2+, Mg2+ and Zn2+ were ineffective. The stimulating effect of Mn2+ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn2+ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn2+) enzyme.
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PMID:Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity. 608 83

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

The catalytic subunit of phosphoprotein phosphatase (Mr = 35,000) is inactivated by phosphate compounds such as trimetaphosphate, PPi, and ATP. The inactivation of phosphoprotein phosphatase by these phosphate compounds is time- and concentration-dependent, is not reversed by dilution or gel filtration and is protected by Pi. A dissociation constant for the enzyme-trimetaphosphate complex and a rate constant for the reaction were calculated to be 4.6 x 10(-4) M and 0.29 min-1, respectively. The inactivation of phosphatase by PPi and ATP shows more complex kinetics than that by trimetaphosphate. The addition of EDTA to PPi and ATP exhibits more potent inactivation, even though EDTA alone does not inactivate phosphatase. This phosphoprotein phosphatase is not labeled by [gamma-32P]ATP. The inactivation of phosphatase by PPi or ATP can only be reversed by Mn2+ or Co2+, among all other metals or cationic compounds tried. The reactivation also requires sulfhydryl compounds. The effectiveness of sulfhydryl compounds follows the order: dithioerythritol greater than mercaptoethanol greater than cysteine. Glutathione was without effect. Metal analysis of the catalytic subunit did not reveal any significant amounts of Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Sn, or Zn. Phosphoprotein phosphatase activity from zinc-deficient rat livers also eliminated the possibility of this phosphatase being a zinc metalloenzyme. Inactivation does not seem to be due to a loss of a critical metal ion. Other mechanisms for inactivation are presented.
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PMID:Inactivation and reactivation of phosphoprotein phosphatase. 627 82

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

The regulation of protein phosphorylation by Zn2+ ions and by other divalent cations was studied in membrane vesicles from a normal mouse epithelial cell line, MMC-E (Mus musculus castaneous). Four major phosphoacceptor polypeptides were found in these membranes. Micromolar concentrations of Zn2+ ions inhibited the phosphorylation of the epidermal growth factor (EGF) receptor and of threonine residues in a 47,000-dalton polypeptide. In contrast, two polypeptides with molecular weights of 54,000 and 57,000 showed increased phosphorylation, mainly of serine residues, in the p.esence of Zn2+ ions. These results were not obtained using similar concentrations of other divalent cations and were apparently not due to an effect of Zn2+ ions on phosphoprotein phosphatases. Thus, the effects of Zn2+ ions on protein phosphorylation in membrane vesicles are complex and are not restricted to an inhibition of a single protein phosphatase or kinase.
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PMID:Effects of Zn2+ ions on protein phosphorylation in epithelial cell membranes. 630 21


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