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)

1. Grinding of epimastigotes of Trypanosoma cruzi with glass powder in a mortar yielded a Mg2+-activated adenosine triphosphatase (ATPase) preparation which was highly sensitive to oligomycin. 2. Chloroform treatment of the particles resulted in the solubilization of an ATPase which was (a) activated by MgCl2; (b) slightly inhibited by CaCl2; (c) activated by sulphite and bisulphite; (d) had an optimum pH of 7.6; and (e) had a Km for ATP of 2.1 mM (in the presence of 4 mM MgCl2). 3. The solubilized enzyme was insensitive to oligomycin and leucinostatin, which inhibited the membrane-bound ATPase, though inhibited by efrapeptin and quercetin. 4. The results indicate that the chloroform-extracted enzyme is a soluble F1-ATPase similar to those isolated from mammalian mitochondria.
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PMID:Solubilization and some properties of the Mg2+-activated adenosine triphosphatase from Trypanosoma cruzi. 16 84

The polypeptide antibiotic duramycin has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al., 1985, Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to probe the role of PE in mitochondrial energy conversion reactions with the following results: (i) Duramycin uncoupled mitochondrial respiration, decreasing the respiratory control ratio to 1 at 5 microM. At concentrations of duramycin in excess of 10 microM, ADP addition inhibited electron transport. (ii) Duramycin inhibited oxidative phosphorylation (C50 less than 2 microM). (iii) Duramycin stimulated mitochondrial ATP hydrolysis modestly. The antibiotic was 7- to 16-fold less effective in this regard than concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (F-CCP) which produced comparable uncoupling. (iv) Duramycin inhibited uncoupled ATPase activity (C50 = 8 microM). Inhibition of the ATPase activity of intact mitochondria was blocked by 1 mM MgCl2 and 5 mM CaCl2; inhibition persisted in sub-mitochondrial particles assayed in the presence of 3 mM MgCl2. The effects on mitochondrial function of free fatty acids (FFA) and duramycin are similar in many respects. It is suggested that duramycin, like FFA, uncouples via a nonclassical mechanism, possibly by disrupting intramembrane H+ transfer between redox and ATPase complexes. In addition, interaction of duramycin, either direct or indirect, with the F0 moiety of the mitochondrial ATPase and with one or more components of the respiratory electron transport chain is proposed.
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PMID:Duramycin effects on the structure and function of heart mitochondria. II. Energy conversion reactions. 165 2

A simple and rapid method for the isolation of bovine heart mitochondrial adenosine 5'-triphosphatase (F1-ATPase) was developed. Mitochondria were purified by differential centrifugation and stored frozen. After thawing. F1-ATPase was released by treatment with chloroform. Purification of the enzyme was achieved by polyethylene glycol precipitation followed by chromatography on Procion Navy H-ER beaded cellulose in the presence of MgCl2. F1-ATPase was eluted by ATP in the absence of MgCl2. The purity of the enzyme was proved by SDS-polyacrylamide-gel electrophoresis. The purified F1-ATPase showed slightly non-hyperbolic kinetics towards ATP and nearly complete inhibition in the presence of millimolar concentrations of ADP.
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PMID:Simple and rapid purification of F1-ATPase from bovine heart mitochondria by affinity chromatography. 183 Apr 76

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].
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PMID:Determination of the roles of active sites in F1-ATPase by controlled affinity labeling. 253 46

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate. 286 80

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.
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PMID:Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion. 289 Mar 77

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.
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PMID:The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase. 289 89

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety. 289 44

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.
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PMID:A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. 290 23

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


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