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)

The energy-rich compound acetyl phosphate (ACP) was examined as a substrate for energy-linked reactions by the yeast plasma membrane H+-ATPase. The hydrolysis of ACP was sensitive to inhibition by vanadate with an IC50 approximately 1 microM, which is comparable to the level obtained in the presence of ATP. A Km of 8.29 +/- 0.65 mM for the hydrolysis of ACP was approximately 10-fold higher than that obtained for ATP, while Vmax values of 8.66 +/- 0.29 and 7.23 +/- 0.34 micromol Pi mg(-1) min(-1) were obtained with ATP and ACP, respectively. ACP formed a phosphorylated intermediate that was efficiently chased with hydroxylamine. Both ACP and ATP effectively protected the enzyme from trypsin-induced inactivation and formed identical tryptic digestion patterns, suggesting that ACP mimics the formation of conformational intermediates induced by ATP. However, unlike ATP, ACP was unable to drive proton transport by H+-ATPase. In addition, a pma1-S368F mutant enzyme that is highly insensitive to inhibition by vanadate in the presence of ATP was largely sensitive to vanadate in the presence of ACP. These results are interpreted in terms of a reverse, short-circuit pathway of the normal P-type ATPase kinetic pathway, in which the formation of E2P by-passes the E1P high-energy intermediate. In this pathway, ACP favors the formation of an E2P conformational state, which can interact with classical inhibitors like vanadate, but possesses insufficient free energy to drive proton transport by the H+-ATPase.
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PMID:Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate. 926 44

The position in the acyl phosphate linkage of the phosphorylated intermediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine was compared with that of the model compound acetylphosphate. The products of the cleavage of the phosphoenzyme by methylhydroxylamine were the active enzyme and a N-P compound, not the inhibited enzyme and inorganic phosphate. This means that the bond cleaved by methylhydroxylamine was the O-P bond, not the C-O bond. In contrast, methylhydroxylamine did not cleave the O-P bond of acetylphosphate in solution, at pH values from 0.3 to 7.0, whether or not the phosphoryl group formed a complex with magnesium. Acetylphosphate and hydroxylamine formed acetohydroxamic acid. Therefore, the state of the acyl phosphate bond in the native phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of their phosphoryl groups or the binding of magnesium to the phosphoenzyme. Molecular orbital calculations for acetylphosphate revealed that the phosphorus atom charge is more positive than the carbon atom, irrespective of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller than that of the C-O bond. Thus, the electronic structure of the acyl phosphate linkage of acetylphosphate under vacuum supports the results obtained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the phosphorylated intermediate of (Na+,K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The phosphoenzyme with bound ouabain, or without a tightly bound divalent cation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.
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PMID:Non-hydrated state of the acyl phosphate group in the phosphorylated intermediate of (Na+,K+)-ATPase. 934

Effect of various inhibitors on the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Ep01(a strain of Amphibacillus xylanus), was examined. Among the chemicals tested, the enzyme was drastically inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate. The ATPase activity of the enzyme, which was inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate, was remarkably restored by beta-mercaptoethanol and hydroxylamine, respectively, suggesting the involvement of cysteine and histidine residues in the enzyme activity. Analysis of the inhibition kinetics by diethyl pyrocarbonate indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme.
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PMID:Possible involvement of cysteine and histidine residues in the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Amphibacillus xylanus. 940 39

Plasma membranes isolated from the marine unicellular alga Tetraselmis (Platymonas) viridis were phosphorylated by [gamma-32P]ATP, and membrane proteins were then analyzed by PAGE in SDS, under acidic conditions. Three radioactive components with apparent molecular masses of 100 kDa, 76 kDa, and 26 kDa were detected. The phosphorylation of one of them, the 100 kDa polypeptide, was specifically stimulated by Na+. Vanadate almost completely inhibited the Na+-mediated phosphorylation of the peptide. The phosphate bound to this peptide underwent rapid turnover and was discharged by hydroxylamine. The 100 kDa phosphopeptide was sensitive to ADP. The conclusion is drawn that the 100 kDa phosphopeptide is a phosphorylated intermediate of the Na+-transporting ATPase in the T. viridis plasma membrane.
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PMID:Na+-ATPase from the plasma membrane of the marine alga Tetraselmis (Platymonas) viridis forms a phosphorylated intermediate. 959 99

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.
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PMID:E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate. 975 50

We purified Cl- pump in the rat brain and obtained 520 or 580 kDa protein complexes which consisted of 62, 60, 55 and 51 kDa proteins. An antiserum against 520 kDa protein complex recognized 51 kDa protein in both 520 and 580 kDa complexes, and reduced both Cl(-)-ATPase and Cl(-) pump activities. Such an immunoreactive 51 kDa protein was found in the brain, spinal cord and kidney. When incubated with [gamma-(32)P]ATP, the protein complex yielded phosphorylated 51 kDa protein, the label being hydroxylamine-sensitive and increased in the presence of Cl- and/or an inhibitor of Cl- pump, ethacrynic acid. Thus, the antibody appears to recognize a possible catalytic subunit of Cl- pump, 51 kDa protein, in the rat.
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PMID:Antiserum against Cl- pump complex recognizes 51 kDa protein, a possible catalytic unit in the rat brain. 987 33

Mouse liver microsomes treated with octylthioglucoside (OTG-microsomes) were examined for copper-stimulated ATPase activity. The activity was about 1 micromol Pi/mg protein/hr under optimal conditions [300 mM KCl, 3 mm MgSO4, 10 mM GSH, 0.5 micron CuSO4, 3 mM ATP and 50 mM acetate buffer at pH5.0]. A reducing agent such as GSH or dithiothreitol was required for the activity, and removal of Cu+ from the reaction mixture by bathocuporinedisulfonate resulted in a complete loss of copper-stimulated ATPase activity. Vanadate inhibited the copper-stimulated ATPase activity. The OTG-microsomes were phosphorylated in a hydroxylamine-sensitive and copper-stimulated way. Iron used instead of copper also stimulated both ATPase and phosphorylation. These results suggest that microsomes from mouse liver contain copper/iron-stimulated P-type ATPase.
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PMID:Characterization of a P-type copper-stimulated ATPase from mouse liver. 1039 56

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.
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PMID:Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule. 1101 56

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na+-K+-ATPase activity as an index. Beta-carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gas-liquid chromatography and high performance liquid chromatography. The Na+-K+-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of beta-carotene oxidative breakdown products, beta-Apo-10'-carotenal and retinal. Most of the beta-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na+-K+-ATPase activity by 50% (IC50) was equivalent to 10 microM non-degraded beta-carotene, whereas the IC50 for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 microM. Carotenoid oxidation products are more potent inhibitors of Na+-K+-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or beta-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.
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PMID:Carotenoid oxidative degradation products inhibit Na+-K+-ATPase. 1102 51

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P<0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.
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PMID:Protein kinase C-induced phosphorylation modulates the Na(+)-ATPase activity from proximal tubules. 1133 27


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