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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.
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PMID:Phosphoester specificity of purified human liver alkaline phosphatase. 3 70

Myosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2,4,6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained. 1. About 0.5 mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP1 (2mM), into both types of myosin studied. 2. The size of the initial burst of P1 liberation for both myosins was found to be 0.5--0.6 mol/mol head. 3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P1 burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst. 4. The EDTA (K+)-ATPase activity of both myosins modified in the presence or absence of PP1 decreased sharply with incorporation of TNBS. 5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10 min of modification with TNBS in the absence of PP1. 6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP1. The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747--753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.
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PMID:Modification of cardiac and smooth muscle myosins with 2,4,6-trinitrobenzenesulfonate. Evidence for differences in structure around the active sites of cardiac, smooth, and skeletal muscle myosin ATPase. 15 5

Rabbit skeletal myosin was trinitrophenylated with 2,4,6-trinitrobenzene sulfonate (TNBS) in the presence or absence of inorganic pyrophosphate (PP1). When myosin trinitrophenylated either in the presence or absence of PP1 was treated with dithiothreitol (DTT), the absorbance at 345 nm of both trinitrophenylated myosins was decreased, as though the trinitrophenyl groups bound to myosin were removed. The DTT treatment also essentially reversed the inhibition of the EDTA-ATPase and Ca-ATPase activities that was caused by trinitrophenylation of myosin. These effects of trinitrophenylation and of DTT treatment were independent of the presence or absence of PP1 during the trinitrophenylation. In contrast, the PP1-induced formation of a difference spectrum of trinitrophenylated myosin was not affected by the DTT treatment. On the basis of these observations, it is suggested that the "reactive lysine residues," trinitrophenylation of which resulted in inhibition of the ATPase activities, are different from those whose trinitrophenyl groups show an altered spectrum on addition of PP1.
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PMID:Trinitrophenylation of rabbit skeletal myosin by 2,4,6-trinitrobenzene sulfonate and treatment of trinitrophenyl myosin with dithiothreitol. 299 Dec 9

The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca2+ elevation in thapsigargin (Tg)-treated platelets and decreased Ca2+ influx into platelets at a time when Ca2+ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn2+) influx (acting as a surrogate for Ca2+ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca2+]i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca2+]i elevation Unexpectedly, PMA inhibited Tg-induced [Ca2+]i elevation in the absence of extracellular Ca2+, and in agreement calyculin A decreased [Ca2+]i elevation almost to basal levels. The results from this study were confirmed with another Ca(2+)-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca2+ influx and in the filling state of the intracellular Ca2+ pool in platelets treated with Tg.
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PMID:A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin. 854 14

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na+-K+-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na+-K+-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific 3H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent 86Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na+-K+-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na+-K+-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na+-K+-ATPases is mediated by a PP2A-dependent dephosphorylation process.
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PMID:Role of protein phosphatase in the regulation of Na+-K+-ATPase by vasopressin in the cortical collecting duct. 884 18

The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.
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PMID:Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease. 1032 4

pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed.
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PMID:The carboxyl-terminal region of the retinoblastoma protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic activity. 1088 4

The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase.
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PMID:Calmodulin effect on purified rat cortical plasma membrane Ca(2+)-ATPase in different phosphorylation states. 1156 65

The transcription factor nuclear factor-kappa-B (NF-kappaB) is now recognised as a key mediator of physiological and pathological plasticity in the central nervous system (CNS), and ionotropic glutamate receptor stimulation potently triggers NF-kappaB activation. This study was designed to identify the mechanisms responsible for the high basal levels of activated NF-kappaB present in neurons in the cerebral cortex. In cultured cortical neurons, the basal levels of activated NF-kappaB were reduced by the glutamate receptor antagonists MK801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but were not affected by exposure to a mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor, a p38 MAP kinase inhibitor or a cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor. However, activated NF-kappaB levels were reduced by a guanylate cyclase inhibitor, the Src-family tyrosine kinase inhibitor PP1, or the farnesyl transferase inhibitors manumycin and farnesyl transferase (Ftase) inhibitor 1. There was no additive effect when MK801 was applied together with manumycin. These results suggest that the basal levels of activated NF-kappaB in cortical neurons are maintained partially by synaptic activity involving N-methyl- D-aspartate (NMDA) and AMPA/kainate glutamate receptors, coupled to activation of an Src-family tyrosine kinase and a p21(Ras)-like guanosine triphosphatase (GTPase) in a cGMP-dependent manner. The results are intriguing in the light of the recent identification of a synaptic p21(Ras) activator stimulated by cGMP.
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PMID:Involvement of NMDA receptors and a p21Ras-like guanosine triphosphatase in the constitutive activation of nuclear factor-kappa-B in cortical neurons. 1242 35

Many cardiac proteins undergo reversible phosphorylation. While the protein kinases which bring about phosphorylations are well studied, less effort has been put into the dephosphorylating phosphatases (for an earlier review compare 14). An important event in the heart, which is controlled by phosphorylation, is the uptake of Ca2+ by the sarcoplasmic reticulum (SR). This process is brought about by a SR Ca2+ ATPase (SERCA) and accounts for relaxation. The amount of Ca2+ pumped by SERCA is enhanced when phospholamban (PLB), an intrinsic protein of the SR, is phosphorylated and is diminished when PLB is dephosphorylated. PLB is dephosphorylated by protein phosphatases (PPs) like PP1. As the activity of PP1 is enhanced in heart failure, subsequent dephosphorylation by of, e.g., PLB may explain the impaired relaxation of the human heart. Thus, PPs may play an important role in the etiology and/or symptoms of heart failure.
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PMID:Altered phosphatase activity in heart failure, influence on Ca2+ movement. 1247 41


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