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)

A Mg2(+)-ATPase-enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anion-exchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore the activity. The Mg-ATPase has a low affinity for Mg2(+)-ATP and is inhibited by fluoride, vanadate, vanadyl and calcium ions. From these characteristics, we infer that this Mg2(+)-ATPase is the same protein as the aminophospholipid translocase which regulates the membrane phospholipid transverse distribution in human erythrocytes by actively transporting aminophospholipids from the outer to the inner monolayer.
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PMID:Partial purification and characterization of the human erythrocyte Mg2(+)-ATPase. A candidate aminophospholipid translocase. 214 4

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.
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PMID:Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles. 214 57

Selective extraction procedure was applied for obtaining different proteins from sarcoplasmic reticulum vesicles following the main steps of previous observations. High concentration of a nonionic detergent, such as polyoxyethylene-10-lauryl ether (C12E10) prevented the formation of Ca(2+)-ATPase crystals. It has been observed that only the 300 kDa protein could induce crystallization from among proteins being undissolved from of membrane. A modification of MacLennan's procedure--applied for ATPase precipitation from deoxycholate solubilized sarcoplasmic reticulum--has been described and an ammonium acetate precipitated Ca(2+)-ATPase was used in the experiments for increase of Ca(2+)-ATPase concentration in the crystallization process. The repeated supplementation of purified and C12E10 solubilized Ca(2+)-ATPase with ammonium acetate precipitated Ca(2+)-ATPase made possible a formation of larger and larger crystals with different periodicity.
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PMID:New approach of three-dimensional crystallization of the Ca(2+)-ATPase of sarcoplasmic reticulum. 215 37

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.
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PMID:Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa. 215 97

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.
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PMID:Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.). 215 72

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.
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PMID:ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein. 215 6

The production of 14CO2 from uniformly labeled glucose was shown to account for the entire increase in histamine-stimulated O2 consumption in rabbit gastric glands when no other substrate was added to the medium. The increased production of CO2 was correlated to the increase in O2 consumption and the accumulation of [14C]-aminopyrine (AP) after stimulation with several secretagogues. Inhibitors of H(+)-K(+)-ATPase reduced the secretagogue-induced increase in CO2 production by greater than 90%, showing that the activity of this enzyme was responsible for the greater part of gastric gland metabolism under stimulated conditions. In contrast to AP accumulation, inhibition of CO2 production by omeprazole, an acid-activated inhibitor of the H(+)-K(+)-ATPase, was not reversed by washing. The reversal of AP accumulation after omeprazole treatment and washing was most likely due to a recruitment of residual pumps bordering a nonacidic space, which had not previously been inhibited by omeprazole. These residual pumps slowly generate a pH gradient and hence AP uptake. Adding NH4+ to gastric glands resulted in a concentration-dependent increase of CO2 production up to the maximal stimulated level but without formation of the pH gradient as measured by AP uptake and loss of the omeprazole inhibition of glucose oxidation. As NH4+ can act as a K+ surrogate for H(+)-K(+)-ATPase, and as NH3 is membrane permeant, full stimulation of CO2 production is evidence that the major mechanism of H(+)-K(+)-ATPase activation in situ is an increase in the KCl permeability of the pump membrane.
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PMID:Coupling of H(+)-K(+)-ATPase activity and glucose oxidation in gastric glands. 215 39

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of Mr 95,000 and Mr 31,000. The genes encoding the large and small subunits (corresponding to the D1 and the D12 ORFs, respectively, of the viral genome) were coexpressed in Escherichia coli BL21 (DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. A 1000-fold purification of the guanylyltransferase was achieved by ammonium sulfate precipitation and chromatography using phosphocellulose and SP5PW columns. Partially purified guanylytransferase synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor. In the presence of AdoMet the enzyme catalyzed concomitant cap methylation with 99% efficiency. Inclusion of S-adenosyl methionine increased both the rate and extent of RNA capping, permitting quantitative modification of RNA 5' ends. Guanylyltransferase sedimented as a single component of 6.5 S during further purification in a glycerol gradient; this S value is identical with that of the heterodimeric capping enzyme from vaccinia virions. Electrophoretic analysis showed a major polypeptide of Mr 95,000 cosedimenting with the guanylyltransferase. RNA triphosphatase activity cosedimented exactly with guanylyltransferase. Methyltransferase activity was associated with guanylyltransferase and was also present in less rapidly sedimenting fractions. The methyltransferase activity profile correlated with the presence of a Mr 31,000 polypeptide. These results indicate that the D1 and D12 gene products are together sufficient to catalyze all three enzymatic steps in cap synthesis. A model for the domain structure of this enzyme is proposed.
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PMID:Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli. 216 22

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle. 216 18

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.
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PMID:Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. 217 11


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