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:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.
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PMID:E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit. 289 2

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.
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PMID:In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri. 292 75

To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO3-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO3-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO3-ATPase in kidney membrane fractions. HCO3-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO3-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO3-ATPase to vanadate indicates that it belongs to the F0-F1 class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO3-ATPase activity at the sites of urinary acidification.
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PMID:Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: localization along the nephron and effect of corticosteroids. 293 49

Thermoacidophilic archaebacteria have gained much interest because of their phylogenetic distance to eubacteria and eukaryotes and also because of their unique living conditions. Investigation of the energy-converting system therefore offers a key for understanding the evolutionary position and environmental adaptation of these unusual bacteria. A plasma-membrane-associated adenosine triphosphatase with specific activities of 0.3-0.6 mumol min-1 (mg protein)-1 has been detected in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639). The enzyme exhibits two optima at pH 5.5 and 8.0, sulfite activation leads to only one optimum at pH 6.25. In the presence of the divalent cations Mg2+ or Mn2+ it hydrolyzes ATP with highest reactivity and also other purine and pyrimidine nucleotides, but not ADP and pyrophosphate. A specific stimulation by monovalent cations is not observed. The ATPase activity is not inhibited by N,N'-dicyclohexylcarbodiimide, azide or vanadate, but it is by the vascular ATPase inhibitor nitrate with an [I]50 of 8 mM. Linear Arrhenius plots up to 75 degrees C reflect pronounced adaptation to the hot environment of the archaebacterium. The solubilized ATPase as localized by activity staining in non-denaturating gels and further analyzed by sodium dodecyl sulfate electrophoresis is composed of two major polypeptides of 65 and 51 kDa reminiscent of the alpha and beta subunits of eubacterial and eukaryotic F0F1-ATPases. The ATPase is suggested as a probable candidate for a reversibly acting ATP synthase responsible for oxidative phosphorylation found in Sulfolobus acidocaldarius.
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PMID:A plasma-membrane associated ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. 295 1

An ATPase co-sedimenting with rat liver peroxisomes has been detected after subcellular fractionation. The activity is Mg2+ dependent, with pH optimum of 7.5 and is inhibited by NEM and DCCD but not by oligomycin. Partial inhibition of the mitochondrial ATPase allows to detect the peroxisomal activity in the gradients. Protease inactivation and solubilization data suggests that the activity resides in a protein of the peroxisomal membrane, exposed to the cytosol.
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PMID:Detection of an ATPase activity in rat liver peroxisomes. 297 19

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.
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PMID:The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni. 297 56

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and this inhibition correlates with incorporation of radioactivity from [14C]DCCD into a Complex I subunit of Mr 29,000 (Yagi, T. (1987) Biochemistry 26, 2822-2828). Resolution of [14C]DCCD-labeled Complex I in the presence of NaClO4 showed that the labeled Mr 29,000 subunit was in the hydrophobic fraction of the enzyme. This fraction, which contains greater than 17 unlike polypeptides, was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the Mr 29,000 subunit, containing bound [14C]DCCD, was isolated and purified. The amino acid composition and partial sequence of this subunit corresponded to those predicted from the mitochondrial DNA for the product of the mtDNA gene designated ND-1. The identity of the Mr 29,000 subunit with the ND-1 gene product was further confirmed by immunoblotting and immunoprecipitation experiments, using the hydrophobic fraction of [14C]DCCD-labeled Complex I and antiserum to a C-terminal undecapeptide synthesized on the basis of the human mitochondrial ND-1 nucleotide sequence. Thus, it appears that the DCCD-binding subunits of the respiratory chain Complexes I, III, and IV and in certain organisms the DCCD-binding subunit of the ATP synthase complex (Complex V) are all mtDNA products.
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PMID:Identification of the dicyclohexylcarbodiimide-binding subunit of NADH-ubiquinone oxidoreductase (Complex I). 314

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.
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PMID:Activation and inhibition of ATP synthesis in cell envelope vesicles of Halobacterium halobium. 358 88

Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about -100 mV relative to the external medium, i.e., inside-negative. ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units. The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about -100 mV. Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N,N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 microM/2 mg protein/ml. These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.
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PMID:ATP synthesis in cell envelope vesicles of Halobacterium halobium driven by membrane potential and/or base-acid transition. 395 92

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.
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PMID:Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria. 609 64


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