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)

A soluble protein phosphatase from the promastigote form of the parasitic protozoan Leishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42,000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+. The pH optimum of the enzyme was 6.5-7.5 and the temperature optimum 37 degrees C. The apparent Km for phosphohistone was 7.14 microM. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest that L. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.
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PMID:Partial purification and characterization of a soluble protein phosphatase from Leishmania donovani promastigotes. 859 23

DNA synthesis was measured 16 h after stimulation of Swiss 3T3 fibroblasts in the resting phase with various growth factors (platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid and thrombin). When extracellular Ca2+ was chelated by EGTA, or when the influx of Ca2+ from outside to inside the cell was blocked by cobalt, DNA synthesis was completely inhibited. As there was no effect whatsoever on DNA synthesis when Ca2+ was chelated, or when the influx of Ca2+ was blocked up to the first 4 h after growth stimulation, it was concluded that, at an early stage, Ca2+ influx from outside to inside the cell is not related to the transition from the G1 to the S phase. A Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) had no effect on DNA synthesis. However, cyclosporin A and FK-506, which are inhibitors of Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin), markedly inhibited DNA synthesis stimulated by all of the growth factors. These results indicate that calcineurin plays a role, not only in activation of T-cells of the immune system in the initial phase, but also in DNA synthesis in fibroblasts. It was concluded that Ca2+ influx from outside to inside the cell during the mid-to-late G1 phase, followed by calcineurin activation, is essential as a mechanism of growth signal transduction.
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PMID:Calcineurin is essential for DNA synthesis in Swiss 3T3 fibroblasts. 876 Mar 49

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg2+ or Mn2+ was essential for maximum phosphorylation although Zn2+, Co2+, and Ca2+ could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.
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PMID:Protein phosphorylation in human peripheral nerve: altered phosphorylation of a 25-kDa glycoprotein in leprosy. 882 44

Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1alpha, was purified in a latent form which was dependent on Co2+ or Mn2+ for activity (Y. Chu, S. E. Wilson, and K. K. Schlender (1994) Biochim. Biophys. Acta 1208, 45-54). This was also true for recombinant PP1 alpha expressed in Escherichia coli (Z. Zhang, G. Bai, S. Deans-Zirattu, M. F. Browner, and E. Y. C. Lee (1992) J. Biol. Chem. 267, 1484-1490). Here we report on the change in the sulfhydryl reactivity during the cation activation process. The activation of myofibrillar PP1 by Co2+ was prevented by 10 mM dithiothreitol (DTT) and incubation of the Co2+-activated enzyme with 50 mM DTT reversed the activation. Activation of recombinant PP1alpha was associated with 57Co2+ incorporation into PP1. DTT reversal of Co2+-activated PP1 was accompanied by release of Co2+ from the enzyme. The latent PP1 modified with 2-nitro-5-thiocyanobenzoic acid (NTCB) or N-ethylmaleimide (NEM) did not bind Co2+ and could not be activated by Co2+. Conversely, the Co2+-activated PP1 was resistant to inactivation with NTCB and less sensitive to NEM. Similarly, PP1 pretreated with NTCB was not activated by Mn2+ and the Mn2+-activated enzyme was also resistant to NTCB inhibition. The number of sulfhydryls of nondenatured PP1, reactive with 5, 5'-dithiobis[2-nitrobenzoic acid] (DTNB), was reduced from approximately 8 to 2-3 mol/mol when the enzyme was activated with Co2+ or Mn2+. After denaturation with guanidine-HCl, the number of reactive sulfhydryls of nonactivated PP1 and Co2+-activated PP1 was approximately 10 mol/mol enzyme. These results suggest that when PP1 is activated by Co2+ or Mn2+, the enzyme undergoes a conformational change resulting in some of the cysteine sulfhydryls no longer being accessible to chemical modification.
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PMID:Effect of activation of protein phosphatase 1 on sulfhydryl reactivity. 883 42

In Swiss 3T3 fibroblasts, growth factor-stimulated progression from G1 to S phase involves activation of the Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (calcineurin). Here we report that both cobalt and the calcium chelator EGTA, inhibitors of calcium uptake, as well as cyclosporin A and FK-506, specific inhibitors of calcineurin function, abolished fibroblast growth factor (FGF)-induced expression of cyclins A and E, but not cyclin D1. At 0.1 microM concentration cyclosporin A completely blocked FGF-induced expression of cyclins E and A and it inhibited FGF-stimulated DNA synthesis by 40%; full inhibition of DNA synthesis required 10 microM cyclosporin A. PD 98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase, and hemicholinium-3, an inhibitor of FGF-induced MAP kinase activity, did not inhibit the stimulatory effect of FGF on the expression of cyclin E. On the other hand, the inhibitory effect of 0.1 microM cyclosporin A on FGF-stimulated DNA synthesis was additive with that of hemicholinium-3, suggesting that the two inhibitors acted by different mechanisms. The inhibitors of calcineurin and calcium uptake also completely blocked the stimulatory effects of lysophosphatidic acid on the expression of cyclins E and A, but not cyclin D1. The results suggest that FGF- or lysophosphatidic acid-induced transcription of cyclin A and cyclin E genes is mediated by calcineurin involving a MAP kinase-independent mechanism and that increased expression of cyclins A and E is required for the maximal stimulatory effects of these mitogens on DNA synthesis.
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PMID:Inhibitors of calcineurin block expression of cyclins A and E induced by fibroblast growth factor in Swiss 3T3 fibroblasts. 960 72

Freshly isolated protein phosphatase 2A (PP2A) was highly active as to the dephosphorylation of protein substrates, but lost most of its spontaneous activity on prolonged storage, and was converted to a latent form requiring Mn2+ or Co2+ ions for activity. In this report, we show that the latent form of PP2A can be activated by the Fe2+/ascorbate system. Activation of the phosphatase required both Fe2+ ions and ascorbate, and the level of activation was dependent on the concentrations of both Fe2+ ions and ascorbate. Both the holoenzyme and catalytic subunit of phosphatase 2A could be activated by the Fe2+/ascorbate system, indicating that direct modulation of the catalytic subunit of the phosphatase by the Fe2+/ascorbate system may cause this activation. Several common divalent metal ions, including Ca2+, Mg2+, Cu2+, Zn2+, and Ni2+ ions, cannot cooperate with ascorbate to activate the phosphatase. Dithiothreitol, a SH-containing reducing agent, could replace ascorbate in the Fe2+/ascorbate system to activate the phosphatase, whereas H2O2, a strong oxidizer, significantly diminished the phosphatase activation by the Fe2+/ascorbate system. The results indicate that iron ions stabilized in the +2 state by reducing agents can activate the phosphatase. Overall, the present study provides initial biochemical evidence suggesting that Fe2+ could be a biologically important metal ion cofactor responsible for PP2A activation.
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PMID:Activation of protein phosphatase 2A by the Fe2+/ascorbate system. 964 67

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase. The complex was inactive and required treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively. The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin. Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1. The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13. 1, 113, and 0.37 nM, respectively. The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels. However, the modest changes are consistent with our previous proposal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7,173-182) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins.
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PMID:Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C mutation. 972 Nov 83

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.
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PMID:Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina. 974 25

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.
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PMID:Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one. 1021 76

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.
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PMID:Effect of substitution inert metal complexes on calcineurin. 1033 77


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