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)

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.
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PMID:The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000. 21 Nov 35

Hyperpolarization of patch-perforated GH3 rat anterior pituitary cells in high-K+ Ca(2+)-free medium reveals an inwardly rectifying K+ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs+, Ba2+, Cd2+ and Co2+, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH3 cell inwardly rectifying K+ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.
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PMID:Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells. 133 77

Nerve growth factor stimulates the uptake of radioactive calcium into PC12 cells. This stimulation is inhibited by low concentrations of dideoxyforskolin or staurosporine, and by high concentrations of nifedipine or cadmium. On the other hand, neither dideoxyforskolin nor staurosporine inhibited the stimulation of calcium uptake caused by BK-8644 or adenosine triphosphate (ATP). Nickel inhibited only the effect of ATP on calcium uptake, and actually stimulated the effects of either BK-8644 or nerve growth factor. Down-regulation of L-calcium channels by BK-8644 blocked the subsequent stimulation of calcium uptake by this agent, but not the stimulation by nerve growth factor. Conversely, pre-treatment of the cells with nerve growth factor inhibited the subsequent stimulation of calcium uptake by nerve growth factor, but not the stimulation by BK-8644. The effects of BK-8644 and nerve growth factor on calcium uptake were additive, as were the effects of nerve growth factor and ATP. Phosphatase 2A inhibited the effect of nerve growth factor on calcium uptake, but did not influence the action of BK-8644. On the other hand, calcineurin inhibited the effect of BK-8644 on calcium uptake, but potentiated the action of nerve growth factor. Calmidazolium or fluphenazine also inhibited the effect of nerve growth factor on calcium uptake, but okadaic acid stimulated it. A comparison of the effects of these inhibitors on the actions of various calcium channel agonists shows that the channels on which the action of nerve growth factor is exerted are different than either the L-type calcium channels or the ATP-activated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nerve growth factor-stimulated calcium uptake into PC12 cells: uniqueness of the channel and evidence for phosphorylation. 137 75

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.
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PMID:Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity. 170 Oct 13

Ultraviolet (280-nm) irradiation of bovine brain calmodulin results in calcium-dependent changes in its fluorescence emission spectrum. These consist of a decline in the intrinsic tyrosine fluorescence of the protein and the appearance of a new emission maximum at 400 nm. Chromatography of irradiated calmodulin, using Ultrogel AcA 54 and phenyl-agarose columns, yields several distinctive fractions. One of these, representing 2.8% of the total recovered protein and 53% of the total fluorescence emission at 400 nm, was selected for detailed characterization. Analyses performed on acid hydrolysates reveal the presence of dityrosine, a derivative of tyrosine known for its fluorescence near 400 nm, at the level of 0.59-0.89 mol per 16,700 g of protein. Sodium dodecyl sulfate gel electrophoresis experiments demonstrate two components of apparent molecular weights 14,000 (80%) and 16,000 (20%). Observations on the effects of UV irradiation on the thrombic fragments of calmodulin and on related calcium binding proteins (rabbit skeletal muscle troponin C, bovine cardiac troponin C, and parvalbumin) support the interpretation that dityrosine formation in calmodulin results from the intramolecular cross-linking of Tyr-99 and Tyr-138. The dityrosine-containing photoproduct of calmodulin is unable to stimulate the p-nitrophenyl phosphatase activity of calcineurin under standard assay conditions. Fluorescence titrations show a generally weakened interaction with calcium ion occurring in two stages. The pKa of the derivative is considerably higher than that of free dityrosine and is calcium dependent, decreasing from 7.88 to 7.59 on the addition of 3 mM CaCl2. Smooth muscle myosin light chain kinase binds the derivative about 280-fold less effectively than it binds native calmodulin. Of several metal ions tested, only Cd2+ approaches Ca2+ in its ability to promote the appearance of the 400-nm emission band during UV irradiation of calmodulin. Mn2+ and Cu2+ appear to inhibit dityrosine formation. Ascorbic acid, dithiothreitol, and glutathione are also inhibitory.
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PMID:Dityrosine formation in calmodulin. 356 41

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

We have detected a protein phosphatase activity in soluble extracts from the halophilic archaeon Haloferax volcanii. This activity was markedly stimulated by the divalent metal ions Mn2+ and Cd2+. It dephosphorylated phosphoseryl residues in casein, mixed histones, and phosphorylase a, but not phosphotyrosyl residues in reduced, carboxyamidomethylated and maleylated lysozyme. This protein phosphatase activity was inhibited by NaF, Zn2+, vanadate, molybdate, inorganic phosphate, inorganic pyrophosphate, or p-nitrophenyl phosphate, or by treatment with diethylpyrocarbonate. Activity was unaffected by other potential inhibitors or activators such as polyamines, heparin, cyclic nucleotides, Ca2+/calmodulin, tartrate, tetramisole, okadaic acid, microcystin LR, or sulfhydryl-modifying agents. The functional similarities between this protein-serine phosphatase and that previously identified in another archaeon, the extreme acidothermophile Sulfolobus solfataricus, suggest the existence of a family of divalent metal ion-stimulated protein-serine phosphatases of extremely ancient origin in the Archaea.
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PMID:A protein-serine phosphatase from the halophilic archaeon Haloferax volcanii. 839 5

Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+]o) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+]o evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2' 2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 microM cAMP was dissolved in the patch pipette solution, high [Ca2+]o still elicited the Cl- current, indicating that the divalent cation-induced Cl- current was carried through Ca(2+)-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl current was mediated through a G protein, and that an increase in [Ca2+]i was critical for the activation of Cl-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca(2+)-activated Cl- channels.
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PMID:Activation of Cl- channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts. 884 38

Xenopus laevis oocytes can be selected to express relatively high levels of endogenous Ca currents. These currents are facilitated by prepulses. Facilitated endogenous Ca currents are unaffected by okadaic acid, RpcAMPS or the dihydropyridine (DHP) antagonist (+) PN 200-110. The endogenous currents and facilitation of endogenous currents by depolarizing prepulses are fully blocked by 1 mM Cd2+. In contrast, oocytes injected with mRNA encoding for the rabbit cardiac alpha 1-subunit express prepulse-facilitated Ca channel currents that are highly enhanced by the phosphoprotein phosphatase inhibitor okadaic acid (3-fold) and blocked by RpcAMPS and the DHP antagonist (+) PN 200-110. While okadaic acid selectively stimulates prepulse facilitation of cardiac alpha 1-subunit Ca currents, the DHP agonist (+) SDZ 202-791 largely increases (5-fold) both the control (before prepulse) and facilitated currents (after prepulse). (+) SDZ 202-791 did not prevent the effect of RpcAMPS or okadaic acid on facilitation of cardiac alpha 1-subunit, suggesting that DHP stimulation is independent of phosphorylation leading to channel facilitation. The enhancement of prepulse facilitation of cardiac alpha 1L-subunit Ca channel current by okadaic acid can be accounted for by a speeding up in the rates of onset during the prepulse. Inhibition of phosphoprotein phosphatases by okadaic acid has only modest effects on the rates of recovery of cardiac alpha 1-subunit Ca channel current from facilitation in the time immediately following the prepulse.
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PMID:Okadaic acid enhances prepulse facilitation of cardiac alpha 1-subunit but not endogenous calcium channel currents in Xenopus laevis oocytes. 902 35

Cadmium, a ubiquitous environmental contaminant, damages several major organs in humans and other mammals. The molecular mechanisms for damage are not known. At high doses (5 mg/kg cadmium chloride or higher), testicular damage in mice, rats, and other rodents includes interstitial edema, hemorrhage, and changes in the seminiferous tubules affecting spermatogenesis. Necrosis is evident by 48 h. The goal of this study was to fine map and identify the cdm gene, a gene that when mutated prevents cadmium-induced testicular toxicity in mouse strains with a mutation in this gene. A serine-threonine phosphatase, calcineurin (CN), subunit A, alpha isoform (Ppp3ca), was one of the seven candidates in the cdm region that was narrowed from 5.6 to 2.0 Mb on mouse chromosome 3. An inhibitor of CN, the immunosuppressant, FK506, prevented cadmium-induced testicular damage in five pathological categories, including vascular endothelial and seminiferous epithelial endpoints. Inductively coupled plasma-mass spectrometry revealed that FK506 protected without lowering the amount of cadmium in the testes. Ppp3ca(-/-) mice were investigated but were found to exhibit endogenous testicular abnormalities, making them an inappropriate model for determining whether the inactivation of the Ppp3ca gene would afford protection from cadmium-induced testicular toxicity. The protection afforded by FK506, found by the current study, indicated that CN is likely to be important in the mechanism of cadmium toxicity in the testis and possibly other organs.
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PMID:FK506, a calcineurin inhibitor, prevents cadmium-induced testicular toxicity in mice. 1778 81


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