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 inhibitory action of synthetic pyrethroids and some chlorinated hydrocarbon insecticides on the neural calcium-calmodulin-dependent protein phosphatase, calcineurin, was studied using one radiotracer and two colorimetric methods. It was found that all insecticidal Type II pyrethroids (cypermethrin, deltamethrin and fenvalerate) are potent inhibitors of isolated calcineurin from bovine brain. Their IC50 values were approximately 10(-9) to 10(-11) M. By contrast, neither noninsecticidal chiral isomers of these pyrethroids, neuroactive Type I pyrethroids nor neuroactive chlorinated hydrocarbon insecticides showed comparable potencies against this enzyme. To confirm the action of Type II pyrethroid in situ, isolated intact rat brain synaptosomes were incubated with [32P]phosphoric acid and subsequently depolarized in the presence and absence of 0.1 microM deltamethrin. As expected, there was a sharp rise in protein phosphorylation due to the action of calcineurin. Deltamethrin caused a distinct delay in the dephosphorylation process. The results clearly indicate that calcineurin is specifically inhibited by Type II pyrethroids.
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PMID:Specific inhibition of calcineurin by type II synthetic pyrethroid insecticides. 131 45

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.
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PMID:A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling. 164 31

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives one protein band (Mr 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (Mr 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 units/mg). However, addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, we have isolated from rat liver a protein that activates the enzyme about 10-fold. This protein has been purified to near homogeneity (Mr 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase does not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 microM) as compared to its action on phosphorylase a (Km = 5.5 microM) and phosphohistone (Km = 20 microM). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain about 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 units/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatase renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. To our knowledge this is the first report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.
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PMID:Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase. 286 Jan 6

The dephosphorylation of rabbit skeletal muscle phosphorylase kinase was studied using two purified rabbit skeletal muscle protein phosphatases. The first enzyme (Mr = 32,000) corresponds to the form we have previously termed protein phosphatase C. Phosphorylase kinase was found to be rapidly dephosphorylated by this enzyme. The site of dephosphorylation was examined, and it was shown that this enzyme was relatively specific for the dephosphorylation of the beta-subunit phosphate, as compared to the alpha-subunit phosphate, of phosphorylase kinase. Phosphate release from the beta-subunit was approximately 100-fold faster than from the alpha-subunit. More importantly, dephosphorylation of the beta-subunit phosphate was not significantly affected by phosphorylation of the alpha-subunit. The dephosphorylation of phosphorylase kinase by a second low molecular weight protein phosphatase, Mr = 33,500, was also studied. The specific activity of this enzyme toward phosphorylase kinase was only a fraction of that exhibited by the Mr = 32,000 phosphatase. This enzyme removed phosphate from both the alpha- and beta-subunits but more rapidly (about 4-fold) from the alpha-subunit. With neither of these enzyme preparations was there any evidence for the regulation of beta-subunit dephosphorylation by phosphorylation of the alpha-subunit as proposed by Cohen and Antoniw ((1973) FEBS Lett. 34, 43-47).
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PMID:Dephosphorylation of rabbit skeletal muscle phosphorylase kinase. Evidence against the operation of the "second-site phosphorylation" mechanism of regulation. 625 53

C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.
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PMID:Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases. 654 9

Potassium transport was measured in equine red blood cells, using 86Rb+ influx as a convenient assay. A significant component of volume- and pH-sensitive K(+)-Cl- cotransport to the overall K+ flux was observed in all blood samples studied, although fluxes were variable between animals, and within individuals when measured at intervals over a period of weeks. The aryloxyacetic acid [(dihydroindenyl)oxy]alkanoic acid (DIOA), at a final concentration of 100 microM, inhibited most (> 95%) of the Cl(-)-dependent K+ flux, and DIOA sensitivity was therefore used to define the activity of the K(+)-Cl- cotransport. K(+)-Cl- cotransport was also sensitive to protein phosphatase inhibition with calyculin A or okadaic acid, with inhibition constants of 9 +/- 1 nM for calyculin and about 100 nM for okadaic acid. Peak fluxes were observed at an external pH of 6.7-7.0, with inhibition at higher and lower values. Volume-sensitive K+ fluxes assayed in autologous plasma, controlled for osmolaity, pH and potassium concentration, were significantly lower (28 +/- 8% of control values, n = 6) than those measured in saline. This inhibition was mimicked by the culture medium RPMI, but disappeared following dialysis of the plasma. Phosphate (5.6 mM) inhibited volume-sensitive K+ fluxes by 48 +/- 2%, n = 3; no significant effect was observed by increasing external magnesium concentrations to 0.5 or 2 mM. Thus, inhibition by RPMI, but not that by plasma, may be due to phosphate. Finally, volume- and pH-sensitive K+ fluxes were sensitive to oxygen tension and were abolished reversibly by equilibrating solutions with nitrogen, as opposed to air. Use of solutions equilibrated with different values of Po2 may account for some of the variability in equine red blood cell KCl fluxes. The importance of these observations to equine red blood cell homeostasis and haemodynamics is discussed.
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PMID:Modulation of K(+)-Cl- cotransport in equine red blood cells. 787 67

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.
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PMID:Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors. 1179 Jan 29

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.
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PMID:In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis. 1237 69

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K(m)) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-A resolution (final R(free) value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to lambda protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.
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PMID:Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase. 1562 11

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P(i)) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P(i) at normal or high external P(i) concentrations) and a high-affinity (activated in response to P(i) starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB(PHO80). We show that the DeltaphoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and DeltaphoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the DeltaphoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1mM P(i). Epifluorescence microscopy revealed accumulation of poly-phosphate in DeltaphoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, DeltaphoB(PHO80) and DeltaphoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis.
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PMID:Functional characterization of the Aspergillus fumigatusPHO80 homologue. 1853 68


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