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 number of approaches were tested for their ability to induce S6 phosphorylation and S6 kinase activation in rat liver, including i.p. injection of insulin, sodium orthovanadate or cycloheximide, as well as refeeding starved animals. All treatments led to increased S6 phosphorylation and activation of the apparent same enzyme. The most potent activator of the S6 kinase in liver extracts was cycloheximide. Maximum activation was achieved in 20 min at 1 mg cycloheximide/100 g body weight, with half-maximal activation in 10 min. Based on these findings a large-scale kinase purification procedure was established involving seven steps of chromatography. Following the final step a major protein band of Mr 70,000 was revealed. The protein was purified 20,000-fold, had a sp. act. of 640 nmol/min/mg of protein towards S6, autophosphorylated and was inactivated by phosphatase 2A. Peptide maps of autophosphorylated material were identical to those derived from the mitogen-activated kinase of 3T3 cells.
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PMID:A stimulated S6 kinase from rat liver: identity with the mitogen activated S6 kinase of 3T3 cells. 268 82

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.
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PMID:Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. 268 46

Treatment of quiescent 3T3 cells with sodium orthovanadate induces a 10-fold stimulation of a kinase that phosphorylates ribosomal protein S6. The kinase in crude extracts is extremely labile and rapidly loses activity when incubated at 37 degrees C. This reaction is blocked by phosphatase inhibitors such as p-nitrophenyl phosphate and beta-glycerophosphate, suggesting that dephosphorylation of the kinase leads to its inactivation (Novak-Hofer, I., and Thomas, G. (1985) J. Biol. Chem. 260, 10314-10319). After three steps of purification the kinase can be separated from greater than 99% of the cellular phosphorylase a phosphatases. At this stage the kinase preparation is almost completely stable but can be inactivated by readdition of specific column fractions that contain both phosphorylase phosphatase and protease activity. However, employing a number of specific inhibitors it is shown that the inactivating agent in these fractions is a protein phosphatase. Furthermore, the physical and enzymatic properties of the kinase inactivator argue that it can be classified as a type 2A phosphatase. These results are consistent with the finding that the purified catalytic subunits of phosphatase type 1 and type 2A also inactivate the kinase. At equivalent phosphorylase a phosphatase activities, the type 2A catalytic subunit is 3 times more potent than the type 1 enzyme in carrying out this reaction. These data indicate that the major S6 kinase inactivator in 3T3 cell extracts is a type 2A phosphatase, supporting the hypothesis that the orthovanadate-stimulated S6 kinase is regulated in vivo by a phosphorylation-dephosphorylation mechanism.
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PMID:Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells. 282 72

The effect of sodium fluoride (NaF) on superoxide generation and cyclic adenosine monophosphate (cAMP) levels in human neutrophils and monocytes was investigated. NaF (greater than 10 mM) stimulated superoxide (O2-) production in both cell types in a time dependent manner. NaF (0.5 to 20 mM) increased cAMP levels by 1.5- to 3.-fold in both neutrophils and monocytes. Increases in cAMP levels were time-dependent; the maximal level was attained within 5 minutes after the addition of NaF, and cAMP levels remained elevated for up to 10 minutes. Only high concentrations of NaF (10 and 20 mM) increased both cAMP levels and O2- production. Therefore, a direct role of cAMP in O2- generation is not likely. It is speculated that since NaF (greater than 10 mM) can complex with extracellular Ca++, and thus reduce free Ca++ concentration required for O2- generation, a NaF-dependent increase in cAMP may restore cytosolic free Ca++ by mobilizing intracellular stores of Ca++. Further, in view of the proposed involvement of a phosphorylation-dephosphorylation mechanism in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, we speculate that NaF, by inhibiting phosphoprotein phosphatase activity, may indirectly activate the NADPH oxidase system and thus superoxide generation.
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PMID:Stimulation of cAMP accumulation and superoxide production in human neutrophils and monocytes. 283

A previous study demonstrated that calcineurin preparations contain variable amounts of endogenous phosphate. This observation suggests that calcineurin may be regulated by protein phosphorylation. In this study we have used calcineurin as a potential substrate for eight different protein kinases and significant phosphorylation was observed only with glycogen synthase (casein) kinase-1 (CK-1). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that only subunit A of calcineurin was phosphorylated. The incorporation of 32P into calcineurin catalyzed by CK-1 ranged from 0.4 to 1.5 mol, depending on the preparation of the substrate used. Peptide mapping revealed that two major sites on calcineurin were phosphorylated. No change in calcineurin activity was observed as a result of phosphorylation. The results of this study suggest that CK-1 may be responsible for phosphorylating calcineurin in vivo.
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PMID:Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1. 283 10

Studies were performed to determine if the Na+-H+ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited 22Na+ uptake in the absence of a pH gradient (pHin 6.0, pHout 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity. These studies indicate that Na+-H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na+-H+ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.
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PMID:Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H+ exchanger. 283 85

Superoxide anion (O2-) is an active oxygen species found in virtually all cells grown in the presence of oxygen. In vivo, the highest concentration of this oxygen radical is found after granulocytes have been exposed to particles or the tumor promoter, phorbol myristate acetate. O2- is released from the cell as a "respiratory burst," which is followed shortly by the appearance of strand breaks in the DNA of the producing cell. In the present report, we have continued our investigation into the mechanism by which extracellular O2- causes breakage of intracellular DNA. Although hydrogen peroxide is present and could also cause strand breaks, its effects are eliminated by the addition of catalase. When the amount of O2- is increased threefold by adding glucose to the medium, the number of breaks increases only slightly, suggesting that the number of breaks that could be induced is limited. The strand-break process is abruptly interrupted by the addition of metabolic poisons such as ionophore A23187, fluoride, or 2-deoxyglucose, but ATP does not appear to be involved. The number of O2(-)-induced strand breaks is increased in the presence of sodium orthovanadate and decreased by A23187. Orthovanadate prevents the inhibition caused by A23187. Reaction of O2- with orthovanadate itself appears not to be responsible for the enhancement of breaks by orthovanadate. We propose that orthovanadate exerts its effect by acting as an inhibitor of a phosphoprotein phosphatase and that A23187 acts to deplete intracellular Ca2+. These data support our hypothesis that the O2- radical causes strand breaks not by attacking the DNA but rather by activating a specific metabolic DNA strand-break pathway.
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PMID:A superoxide anion induced DNA strand-break metabolic pathway in human leukocytes: effects of vanadate. 284 51

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.
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PMID:Purification and properties of the phosphorylated form of guanylate cyclase. 289 12

The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase.
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PMID:Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions. 291 40

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.
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PMID:Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover. 291 46


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