Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.
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PMID:Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan. 252 17

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.
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PMID:Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. 285 69

The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.
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PMID:Studies on Mg2+-dependent ATPase in bovine adrenal chromaffin granules. With special reference to the effect of inhibitors and energy coupling. 288 84

Isolation of F1-ATPase from Rhodospirillum rubrum by chloroform extraction of chromatophores, followed by purification on a glycerol gradient, results in a very pure enzyme preparation containing five subunits with high Ca2+-ATPase activity (15 mumol per min per mg protein). Furthermore, conditions are reported under which the purified F1 exhibits Mg2+-dependent ATPase activity of about 35 mumol per min per mg protein. NaHCO3 stimulates the Mg2+-activity from 1.5 mumol per min per mg protein to 5 mumol per min per mg protein giving a maximal activity at a concentration of about 60 mM NaHCO3. Lauryl dimethylamine oxide (LDAO), octyl glucoside and nonanoyl N-methylglucamide enhance the Mg2+-ATPase activity from 1.5 to 14, 22 and 35 mumol per min per mg protein, respectively, in the absence of NaHCO3, and from 5 to 34, 30 and 37 mumol per min per mg protein, respectively, in the presence of 50 mM NaHCO3. The Vmax is increased, but the Km for ATP remains the same, about 0.22 mM, both in the absence of activators and in the presence of NaHCO3, LDAO or NaHCO3 plus LDAO. Ca2+-dependent ATPase activity is slightly stimulated by NaHCO3 but strongly inhibited by octyl glucoside.
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PMID:Conversion of coupling factor 1 of Rhodospirillum rubrum from a Ca2+-ATPase into a Mg2+-ATPase. 290 Dec 72

The hydrophobic compound diethylstilbestrol inhibits the generation of the proton gradient and the membrane potential in chromatophores from Rhodospirillum rubum and dissipates proton gradients over asolectin vesicle membranes. The Ca2+-ATPase activity of chromatophores, of purified F0F1-ATPase and of purified F1-ATPase is also decreased in the presence of diethylstilbestrol. Other repressed activities are the pyrophosphatase activity of soluble pyrophosphatase from yeast and the NADH oxidation by L-lactate:NAD oxidoreductase. We have previously reported that also ATP synthesis, PPi synthesis and PPi hydrolysis of R. rubrum chromatophores are inhibited by diethylstilbestrol [Strid et al. (1987) Biochim. Biophys. Acta 892, 236-244]. Addition of bovine serum albumin reverses or prevents diethylstilbestrol-induced inhibition of the activities tested. On the other hand, the Mg2+-ATPase activity of chromatophores, purified F0F1-ATPase and purified F1-ATPase are stimulated by low concentrations of diethylstilbestrol. On the basis of its hydrophobicity and the reversal of its inhibition by bovine serum albumin, diethylstilbestrol is proposed to act unspecifically on membranes and at hydrophobic domains of proteins. Such an attack upon the subunits of the F1-ATPase, altering the subunit interactions, is proposed to explain the different results obtained for the Ca2+-ATPase and the Mg2+-ATPase.
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PMID:Diethylstilbestrol. Interactions with membranes and proteins and the different effects upon Ca2+- and Mg2+-dependent activities of the F1-ATPase from Rhodospirillum rubrum. 290 53

The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented.
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PMID:Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes. 614 98

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na+ + K+)-ATPase (ouabain-sensitive), Mg2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPases, for example 7.9 and 10 mM tetracaine for Mg2+-ATPase and (Na+ + K+)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.
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PMID:Inhibition of synaptosomal enzymes by local anesthetics. 614 63

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the alpha-subunit (52 kDa) and beta-subunit (50 kDa) of electric organ mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido-ATP shows a 50-kDa band. Vesicle ghosts were found to accumulate [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump. We conclude that cholinergic vesicles contain a proton pump probably driven by the Mg2+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.
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PMID:Characterization of a Mg2+-ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata. 614 23

Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.
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PMID:EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168). 1006 66

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.
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PMID:Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles. 1829 42


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