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)

The lipid composition of yeast cells was manipulated by the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. There was a 2-3-fold decrease in the concentration of cytochromes a+a3 when the unsaturated fatty acid content of the cells was decreased from 60-70% of the total fatty acid to 20-30%. The amounts of cytochromes b and c were also decreased under these conditions, but to a lesser extent. Further lipid depletion, to proportions of less than 20% unsaturated fatty acid, led to a dramatic decrease in the content of all cytochromes, particularly cytochromes a+a3. The ATPase (adenosine triphosphatase), succinate oxidase and NADH oxidase activities of the isolated mitochondria also varied with the degree of unsaturation of the membrane lipids. The lower the percentage of unsaturated fatty acid, the lower was the enzymic activity. Inhibition of mitochondrial ATPase by oligomycin, on the other hand, was not markedly influenced by the membrane-lipid unsaturation. Npn-linear Arrenius plots of mitochondrial membrane-bound enzymes showed transition temperatures that were dependent on the degree of membrane-lipid unsaturation. The greater the degree of lipid unsaturation, the lower was the transition temperature. It was concluded that the degree of unsaturation of the membrane lipids plays an important role in determining the properties of mitochondrial membrane-bound enzymes.
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PMID:Membrane-lipid unsaturation and mitochondrial function in Saacharomyces cerevisiae. 12 85

The phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins. We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids. The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of alpha-helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition. The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics. The conformational effect of anesthetics is not directly exerted on the proteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition.
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PMID:Biophysical studies on agents affecting the state of membrane lipids: biochemical and pharmacological implications. 15 58

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.
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PMID:Properties of membranes from mutant strains of Escherichia coli in which the beta-subunit of the adenosine triphosphatase is abnormal. 15 58

1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.
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PMID:The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration. 16 20

The effect of temperature on the activation energies of mitochondrial enzymes of the yeast Saccharomyces cerevisiae was examined. Non-linear Arrhenius plots with discontinuities in the temperature range 14-19 degrees C and 19-22 degrees C were observed for the respiratory enzymes and mitochondrial ATPase (adenosine triphosphatase) respectively. A straight-line Arrhenius plot was observed for the matrix enzyme, malate dehydrogenase. The activation energies of the enzymes associated with succinate oxidation, namely, succinate oxidase, succinate dehydrogenase and succinate-cytochrome c oxidoreductase, were in the range 60-85kJ/mol above the transition temperature and 90-160kJ/mol below the transition temperature. In contrast, the corresponding enzymes associated with NADH oxidation showed significantly lower activation energies, 20-35kJ/mol above and 40-85kJ/mol below the transition temperature. The discontinuities in the Arrhenius plots were still observed after sonication, treatment with non-ionic detergents or freezing and thawing of the mitochondrial membranes. Discontinuities for cytochrome c oxidase activity were only observed in freshly isolated mitochondria, and no distinct breaks were observed after storage at -20 degrees C. Mitochondrial ATPase activity still showed discontinuities after sonication and freezing and thawing, but a linear plot was observed after treatment with non-ionic detergents. The results indicate that the various enzymes of the respiratory chain are located in a similar lipid macroenvironment within the mitochondrial membrane.
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PMID:Phase transitions in yeast mitochondrial membranes. The effect of temperature on the energies of activation of the respiratory enzymes of Saccharomyces cerevisiae. 16 75

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.
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PMID:ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity. 17 67

Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.
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PMID:Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III) 41 53

Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.
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PMID:Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart. 132 29

We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that oxidative phosphorylation is often close to equilibrium (apart from cytochrome oxidase) and as a consequence respiration and ATP synthesis are mainly regulated by (a) the phosphorylation potential, and (b) the NADH/NAD+ ratio. However, oxidative phosphorylation is not always close to equilibrium, at least in isolated mitochondria, and relative proximity to equilibrium does not prevent the respiratory chain, the proton leak, the ATP synthase and ANC having significant control over the fluxes. Thus in some conditions respiration rate correlates better with [ADP] than with phosphorylation potential, and may be relatively insensitive to mitochondrial NADH/NAD+ ratio.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of respiration and ATP synthesis in mammalian mitochondria and cells. 159 89

Rotenone-sensitive, uncoupler-insensitive, NADH-dependent respiration was demonstrated in osmotically inactive fragments of the mitochondrial inner-membrane obtained following high amplitude (spontaneous) swelling. This NADH-dependent respiration as well as mitochondrial ATPase activity was stimulated by ligands which are known to be transported by specific transporters/mechanisms. The ligands capable of this anomalous respiratory control included several intermediates of the citric acid cycle, besides non-metabolizable ligands including lactate, cations such as K+ and Ca2+. The interaction between NADH-dependent respiration and these ligands, as manifested by stimulation of respiration, was strongly ionic strength-dependent. The thermodynamic relationship between respiratory control and stimulation of transport ATPase by the relevant transportable ligands could also be demonstrated in the conventional (rat liver) microsomes. These experimental results offer a novel experimental base for search into an intra-membranous mechanism of energy transduction.
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PMID:NADH-dependent respiration in osmotically inactive swollen mitochondria: does transport replace phosphorylation in mediating respiratory control in swollen mitochondria? 175 31


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