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 ATPase of Bacillus alcalophilus was extracted from the bacterial membranes with Triton X-100 and purified by hydroxyapatite column chromatography. SDS gel-electrophoresis of the purified protein indicated the typical subunit pattern of an F1F0 structure with five F1 subunits (alpha, beta, gamma, delta, epsilon) and three F0 subunits (a,b,c). The alpha and beta subunits were antigens for an antiserum against the corresponding subunits of the ATPase of Escherichia coli. Subunit c was extracted from the bacterial membranes with chloroform/methanol. Its amino acid composition was in the range of subunits c from other ATPases. Maximal ATPase activity was observed in the presence of 2-5 mM MgCl2, an ATP/Mg2+ ratio of 2:1 and 25% methanol. In the absence of methanol, only about 1% of the maximal activity was observed. The enzyme was also activated by Ca2+ (in the absence of methanol), reaching about 30% of the maximal activity. The dependence of initial velocity versus ATP of the Ca2(+)-activated but not of the Mg2+/methanol-activated enzyme indicted cooperativity with three strongly cooperative binding sites.
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PMID:The ATPase of Bacillus alcalophilus. Purification and properties of the enzyme. 214 15

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 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

1. Suramin, an inhibitor of several types of ATPase, was investigated for its ability to antagonize responses mediated via P2X-purinoceptors in the guinea-pig urinary bladder and P2Y-purinoceptors in the guinea-pig taenia coli. 2. In isolated strips of bladder detrusor muscle, suramin (100 microM-1 mM) caused a non-competitive antagonism of responses to alpha, beta-methylene ATP with an estimated pA2 of approximately 4.7, and inhibited responses to stimulation of the intramural purinergic nerves, with a similar pA2 value. At a concentration of 10 microM, suramin had little effect, but at a concentration of 1 microM, suramin potentiated responses to alpha,beta-methylene ATP, and potentiated responses to electrical stimulation of intramural purinergic nerves. 3. In isolated strips of taenia coli, in which a standard tone had been induced by carbachol (100 nM), suramin at 100 microM and 1 mM significantly antagonized relaxant responses to ATP (at an EC50 concentration) with an estimated pA2 of 5.0 +/- 0.82 and relaxant responses to electrical stimulation of the intramural non-adrenergic, non-cholinergic inhibitory nerves, either single pulses or trains of 8 Hz for 10 s, with estimated pA2 values of 4.9 +/- 0.93 and 4.6 +/- 1.01, respectively. Suramin had no significant effect at 1 or 10 microM. 4. Suramin, at any of the concentrations tested, did not affect contractile responses to histamine (10 microM) or carbachol (10 microM) in the bladder detrusor preparations. In the taenia coli, suramin did not affect either the relaxant responses to noradrenaline (at an EC50 concentration) or the contractile responses to carbachol (100 nM). 5. Thus, suramin at concentrations above 10 microM blocked actions mediated via P2x- and P2y-purinoceptors in the guinea-pig urinary bladder and taenia coli respectively. Potentiation of purinoceptor-mediated activity was seen only at a low concentration of suramin (1 microM) and only in the urinary bladder (P2x-purinoceptor). For its antagonistic activity suramin did not discriminate between P2X- and P2y-purinoceptors, but it was selective for P2-purinoceptor-mediated activity rather than that mediated via cholinoceptors, adrenoceptors or histamine receptors.
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PMID:Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. 233 85

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.
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PMID:Purification and characterization of primer recognition proteins from HeLa cells. 236 57

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.
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PMID:Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity. 241 24

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.
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PMID:Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport. 242 57

The synthesis and assembly of chloroplast H+-ATPase complex were studied by analyzing the incorporation of [35S]methionine into the constituent subunits with isolated intact chloroplasts and with thylakoid membranes that had been prepared from the chloroplasts so that they would retain ribosomes. The complex was isolated from thylakoids after labeling and identified by immunoprecipitation with an antiserum specific to CF1. The mechanism for the assembly of the complex was demonstrated to be active in the isolated chloroplasts by the following observations: the plastid genome-regulated subunits (alpha, beta, epsilon, I, and III) were labeled by in organello translation and recovered with the complex, and three other subunits (gamma, delta, and II) were labeled when intact chloroplasts were incubated with translation products from polyadenylated RNA. The two largest subunits, alpha and beta, were translated on thylakoid-bound ribosomes when the thylakoid membranes were incubated with soluble factors from Escherichia coli. They were recovered with the H+-ATPase complex, suggesting that they are translated on the bound ribosomes in the chloroplast, and that the isolated membranes retain the ability to assemble a complete complex. Provided that these observations are the result of de novo assembly of the complex, the imported and processed nuclear-coded subunits are presumed to be pooled not in stroma but on the membrane.
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PMID:Synthesis and assembly of H+-ATPase complex by isolated "rough" thylakoids. 244 27

Monoclonal antibodies directed against epitopes on each of the five subunits (alpha, beta, gamma, delta, and epsilon) of the Escherichia coli F1 ATPase (ECF1) have been prepared and used to localize the subunits in the enzyme complex. Fab' fragments, prepared by pepsin digestion of the antibodies, were bound to ECF1 and visualized by cryoelectron microscopy of the unstained, frozen hydrated ECF1-Fab' complexes. Besides aiding in the identification of the ECF1 subunits, addition of Fab's to the specimen fortuitously offers additional advantages in this technique. ECF1 labeled with anti-alpha Fab' is uniformly oriented in the amorphous ice layer, in contrast to unlabeled ECF1, which exhibits a multitude of projection views when examined in ice. Almost all complexes display a triangular projection, which image averaging reveals to be a hexagonal view of ECF1 with Fab' fragments labeling every other peripheral subunit, confirming the alternating arrangement of alpha and beta subunits in the enzyme. A density in the interior of the structure is positioned asymmetrically, adjacent to an unlabeled peripheral mass, indicating that its primary linkage is to a beta rather than an alpha subunit. The composition of the asymmetric density was explored by examining the trypsin-treated ECF1, taking advantage of the unique orientation induced by the binding of anti-alpha Fab'. Trypsin treatment releases the delta and epsilon subunits and cleaves the gamma subunit; the internal density is reduced but not eliminated, showing the contribution of the gamma subunit to the residual structure, and suggesting that the loss of the delta and epsilon subunits, or a structural rearrangement of the gamma subunit, is responsible for its smaller size.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cryoelectron microscopy of Escherichia coli F1 adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex. 247 70

Mutants of Vibrio parahaemolyticus lacking the H+-translocating ATPase were isolated to evaluate both the role of this enzyme and the possibility of the involvement of other cation-translocating ATPase in the energy transduction in this organism. Dicyclohexylcarbodiimide-sensitive ATPase activity which represents the H+-translocating ATPase was not detected either in the membrane vesicles or in the cytosol of the mutants. Three major subunits, alpha, beta and gamma, of the H+-translocating ATPase were missing in the membranes of the mutants. Although ATP was synthesized in wild type cells when an artificial H+ gradient was imposed, little ATP was synthesized in the mutants. However, we observed a large ATP synthesis driven by the respiration not only in the wild type but also in the mutants. The respiratory-driven ATP synthesis in wild type was inhibited by an H+ conductor, carbonylcyanide m-chlorophenylhydrazone, by about 50%. On the other hand, the ATP synthesis in the mutants was not affected by the H+ conductor. Since this organism possesses a respiratory Na+ pump, Na+-coupled ATP synthesis might take place. In fact, we observed some ATP synthesis driven by an artificially imposed Na+ gradient both in the wild type and the mutant.
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PMID:A respiratory-driven and an artificially driven ATP synthesis in mutants of Vibrio parahaemolyticus lacking H+-translocating ATPase. 252 19


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