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)

Bovine heart mitochondrial ATPase is inhibited after covalent modification with 4-chloro-7-nitrobenzofuroxan. The kinetics of the reaction are indistinguishable from those for the reaction of an essential tyrosine residue of the ATPase with 4-chloro-7-nitrobenzofurazan that have been described previously [Ferguson et al. (1975) Eur. J. Biochem. 54, 117-126]. 4-Fluoro-7-nitrobenzofurazan inhibits the ATPase with a pseudo-first-order rate constant that is tenfold greater than that for 4-chloro-7-nitrobenzofurazan. These data indicate that the rate-limiting step for reaction of the enzyme with these reagents is formation of a Meisenheimer complex at the C-4 position and that the modified tyrosine is probably on the surface of the protein. No evidence was found for more complex patterns of reactivity of 4-chloro-7-nitrobenzofurazan and its analogues. Both ammonium 4-chloro-7-sulphobenzofurazan and ammonium 4-fluoro-7-sulphobenzofurazan fail to react with the ATPase. The utility of these reagents as alternatives to the nitro derivatives may be limited owing to their slow reaction rates. After modification on tyrosine by 4-chloro-7-nitrobenzofurazan, the nitrobenzofurazan group can be transferred by an intramolecular process to lysine [Ferguson et al. (1975) Eur. J. Biochem. 54, 127-133]. ATPase with the lysine thus modified is shown to be reactive towards 4-chloro-7-nitrobenzofurazan in a manner indistinguishable from the native enzyme. This indicates that the intramolecular transfer occurs at sufficient distance to avoid steric hindrance to the second reaction, and that the lysine does not participate in a neighbouring group effect to enhance the reactivity of the tyrosine.
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PMID:The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4-chloro-7-nitrobenzofurazan and related compounds. 623 12

Mitochondrial F1-ATPase from beef heart, forms aggregates when it is depleted of loosely bound nucleotides by repeated precipitation in ammonium sulfate. Polyacrylamide gradient gel electrophoresis, in non dissociating conditions shows that the aggregate formed is a dimer (708,000 daltons). The aggregation is attributed to a conformational change of the protein as a consequence of the elimination of the nucleotides from the low affinity binding sites. This structural alteration seems to be reversible because, after addition of ATP, the aggregation is not observed on polyacrylamide gels but the catalytic properties remain unchanged. This conformational change alters the accessibility of protein sulfhydryl groups to 5,5' - dithiobis(2-nitrobenzoic acid). All these observations emphasize the importance of protein nucleotide interactions to the conformation of the mitochondrial F1-ATPase.
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PMID:Structural changes in mitochondrial F1-ATPase induced by the removal of loosely bound nucleotides. 623 25

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.
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PMID:Structural rearrangements in soluble mitochondrial ATPase. 645 13

The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The 32Pi-ATP exchange activity of the reconstituted ATPase complex was inhibited by p-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with 14C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of 32Pi-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved: at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of 32Pi-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.
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PMID:Isolation, characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump. 646 Jul 56

The methyl 4-azidobenzimidate derivative of the naturally occurring ATPase inhibitor protein (IF1) of mitochondria binds to the beta subunits of soluble F1-ATPase upon photoactivation [Klein, G., Satre, M., Dianoux, A.-C., & Vignais, P. V. (1981) Biochemistry 20, 1339--1344]. A number of specific ATPase inhibitors, namely, 4-chloro-7-nitrobenzofurazan (NBF-Cl), efrapeptin, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), phenylglyoxal, aurovertin, tridentate ferrous bathophenanthroline, and octylguanidine (referred to hereafter as "artificial" inhibitors), are also considered to bind to the beta subunit, and there is strong evidence that the first three bind at the active site. Since the inhibition by IF1 of complex V ATPase activity can be reversed by incubation of the inhibited complex at pH 8.0, this system was used to investigate whether the inhibitions brought about by IF1 and the artificial inhibitors were independent, mutually interfering, or mutually exclusive. The experiments were carried out in two ways. (a) Complex V was first maximally inhibited by IF1. Then an artificial inhibitor was added and allowed to react. Excess artificial inhibitor was removed by precipitation of the doubly inhibited complex V with ammonium sulfate and resuspension in inhibitor-free buffer at pH 8.0. Incubation at pH 8.0 released the inhibition due to IF1. However, it was found that the factor that controlled reemergence of ATPase activity was the degree of inhibition exerted by the artificial inhibitor. When the artificial inhibitor was removed first (which was done by addition of dithiothreitol when the artificial inhibitor was NBF-Cl), then reemergence of activity depended on incubation at pH 8.0 to reverse the inhibition due to IF1. These results indicated that IF1-inhibited complex V could be independently inhibited by various artificial inhibitors. The artificial inhibitors used in this type of study were NBF-Cl, efrapeptin, aurovertin, FSBA, and phenylglyoxal. (b) Complex V was first treated with the artificial inhibitor (ferrous bathophenanthroline or octylguanidine) and then with IF1. Results showed that prior treatment of complex V with these inhibitors did not interfere with IF1 subsequently exerting maximal and reversible inhibition. The above results have been discussed in view of the recent finding that F1-ATPase contains two functional and interacting hydrolytic sites [Grubmeyer, C., & Penefsky, H.S. (1981) J. Biol. Chem. 256, 3718--3727].
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PMID:Independent inhibitions of mitochondrial complex V by the adenosinetriphosphatase inhibitor protein and active-site modifiers. 646 71

Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The alpha- and beta-subunits of F1-ATPase were major components of the preparation.
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PMID:Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane. 711 99

ATP induced swelling of isolated yeast mitochondria suspended in an isoosmotic solution of potassium gluconate. Valinomycin stimulated the swelling rate, indicating that K+ influx in the presence of ATP is rate-controlling. This swelling was inhibited by ADP, phosphate (probably acting on the external face of the inner membrane), and Mg2+, which forms a complex with ATP. ATP-induced swelling did not require working F0-F1-ATPase since it was not inhibited by oligomycin and uncoupler. CTP and GTP also induced a swelling. ATP also induced mitochondrial swelling in potassium glutamate, chloride, and acetate but not in phosphate solutions. Sodium, but not ammonium, can replace potassium ion. It is probable that the ATP-channel opening also necessitates an electrogenic cation influx. Respiration also induced swelling of mitochondria suspended in isoosmotic potassium gluconate solution. ATP- or respiration-induced swelling were inhibited equally by N,N'-dicyclohexylcarbodiimide, propranolol, and Zn2+ but not by quinine; all these drugs inhibit the H+/K+ exchange. It was concluded that this unspecific channel is not open under conditions used to measure oxidative phosphorylation. Its physiological role remains unknown.
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PMID:ATP-induced unspecific channel in yeast mitochondria. 752 86

The mutant beta subunit of F1-ATPase from a thermophilic Bacillus strain, PS3, in which tyrosine at position 341 is replaced by leucine (beta Y341L) was expressed in Escherichia coli and crystallized by the vapor-diffusion procedure. Small needle-like crystals were obtained using ammonium sulfate as a precipitant and grown by the stepwise seeding method. The crystals obtained by this procedure diffracted X-rays to about 3 A resolution. The diffraction patterns indicated that the crystals belong to the orthorhombic system and the space group I222 or I2(1)2(1)2(1) with unit-cell dimensions of a = 232 A, b = 66 A, and c = 80 A. It is thought that the asymmetric unit comprises one beta Y341L molecule.
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PMID:Crystallization of mutant beta subunit of F1-ATPase from thermophilic Bacillus PS3. 793 28

Recent data showed storage of subunit c of mitochondrial ATP synthase in late infantile, juvenile, and adult forms of neuronal ceroid lipofuscinosis (NCL). The present study demonstrates that the expression of subunit c in NCL fibroblasts in long-term cultures, both grown in standard conditions and after leupeptin and ammonium chloride treatment, is not greater than in controls. It indicates that as a result of yet undefined factors, NCL fibroblasts in long-term cultures, lose their ability to accumulate subunit c. Moreover, both Western blot analysis of brain tissue homogenates and immunohistochemistry showed increased immunoreactivity to subunit c in mucopolysaccharidosis type I and III. This increased subunit c expression in a disorder with impaired lysosomal function other than the NCL supports the hypothesis that accumulation of this proteolipid might be related to its defective degradation.
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PMID:Increased expression of subunit c of mitochondrial ATP synthase in brain tissue from neuronal ceroid lipofuscinoses and mucopolysaccharidosis cases but not in long-term fibroblast cultures. 815 85

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.
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PMID:F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase. 824 Feb 95


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