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PubMed1979

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.

Adenosine Triphosphatases↗
PubMed1980

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+.

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.

Adenosine Triphosphatases↗
PubMed1981

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria.

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K+. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K+-depleted mitochondria induced by the polyether is reversed by concentrations of K+ higher than 60 mM, and also by Li+, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K+ at high concentrations, and Li+. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.

Animals↗
PubMed1982

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane.

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.

Adenosine Triphosphate↗
PubMed1983

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of delta pH in mitochondrial ATPase-ATP synthase.

Using fluorescence quenching of 9-amino-6-chloro-2- methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlation have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of delta pH. The results obtained are best explained in terms of local circuits of protons.

ATP Synthetase Complexes↗
PubMed1984

Substrate-site interactions in the membrane-bound adenine-nucleotide carrier as disclosed by ADP and ATP analogs.

The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid. The nucleotides tested fell into two classes depending on the shape of the binding curve. Curvilinear Scatchard plots were obtained for the binding of ADP, ATP, adenosine 5'-triphospho-gamma-1-(5-sulfonic acid)naphthylamidate [gamma-AmNS)ATP) and adenylyl (beta,gamma)-methylenediphosphate (p[CH2]ppA); on the other hand, rectilinear Scatchard plots were obtained in the case of naphthoyl-ADP (N-ADP) and 8-bromo ADP (8Br-ADP) binding. The total number of binding sites for N-ADP and 8Br-ADP could be extrapolated with good accuracy to 1.3-1.5 nmol/mg protein; this value corresponds to the number of carboxyatractyloside-binding sites in heart mitochondria (Block, M.R., Pougeois, R. and Vignais, P.V. (1980) FEBS Lett. 117, 335-340). On the other hand, because of the curvilinearity of the Scatchard plots for the binding of ADP, ATP, (gamma-AmNS)ATP and p[CH2]ppA, the total number of binding sites for these nucleotides could only be approximated to a value higher than 1 nmol/mg protein, the exact value being probably equal to that found for N-ADP and 8Br-ADP binding, i.e. 1.3-1.5 nmol/mg protein. Curvilinearity of Scatchard plots was discussed in terms of negative interactions between nucleotide-binding sites located on the same face of the adenine nucleotide carrier. A possible relationship between the features of the binding plots and the transportable nature of the nucleotide is discussed. Contrary to the enhancing effect of bongkrekic acid on [14C]ADP uptake observed essentially in nucleotide-depleted heart mitochondria (Klingenberg, M., Appel, M., Babel, W. and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654), binding of bongkrekic acid to nondepleted heart mitochondria was found to partially displace previously bound [14C]ADP. These opposite effects of bongkrekic acid may be explained by assuming that bongkrekic acid is able to abolish negative cooperativity between external (cytosolic) ADP-binding sites.

Adenosine Diphosphate↗
PubMed1984

Inhibition of mitochondrial oxidative phosphorylation by 2-methyl-4-dimethylaminoazobenzene.

The azodye 2-methyl-4-dimethylaminoazobenzene inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. Phosphorylation was more sensitive to the inhibitory action of the azodye than was the oxidation of succinate or ascorbate. The oxidation of NAD+-linked substrate was severely inhibited by the compound. In submitochondrial particles, only NADH oxidation was sensitive. The site of inhibition has been identified to lie between the dehydrogenase flavoprotein and ubiquinone.

Animals↗
PubMed1984

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex.

Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-Pi exchange activity. Aurovertin alone inhibits 50% of the oxonol shift at 2 microM, and no further inhibition occurs at up to 12 microM. In combination with even weakly effective uncouplers, 4 microM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10-27%, depending on the preparation. The extent of the oxonol response depends on the ATP/ADP ratio rather than the phosphorylation potential. The dye response in the ATPase complex is 4-7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.

Animals↗
PubMed1985

Orientation of cytochrome c oxidase molecules in the two populations of reconstituted vesicles resolved by column chromatography on DEAE-Sephacryl.

Vesicles reconstituted with bovine heart cytochrome c oxidase and dioleoylphosphatidylcholine can be resolved into two populations by column chromatography in DEAE-Sephacryl (Madden, T.D. and Cullis, P.R. (1984) J. Biol. Chem. 259, 7655-7658). These two fractions (I and II) were treated with two proteases. These are trypsin, which has been found to cleave subunit IV in the M domain of the cytochrome c oxidase molecule, and chymotrypsin, which has been found to cleave subunit III in the C domain. These studies show that fraction I vesicles contain cytochrome c oxidase orientation with the M domain outside, i.e., in the same topology as in submitochondrial particles, while fraction II vesicles contain enzyme molecules with their C domain outside, and thus in the same orientation as in mitochondria.

Animals↗
PubMed1985

Coupling factor activity of the purified pea mitochondrial F1-ATPase.

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.

Adenosine Triphosphate↗
PubMed1987

Reaction of electron-transfer flavoprotein ubiquinone oxidoreductase with the mitochondrial respiratory chain.

Submitochondrial particles catalyze the reduction of electron-transfer flavoprotein (ETF) by NADH and succinate under anaerobic conditions in reactions that are totally inhibited by rotenone and thenoyl trifluoroacetone, respectively. The particles also catalyze the ATP-dependent reduction of NAD+ by enzymatically reduced ETF. The latter reaction is inhibited by rotenone and carbonyl cyanide chlorophenylhydrazone and all three reactions are inhibited by antibody to electrontransfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). These observations indicated that ETF-QO reacts with the pool of ubiquinone that is reduced by NADH and succinic dehydrogenases. Consistent with this hypothesis, NADH- and succinic-ETF reductase activities are inhibited 99% in ubiquinone-depleted particles, and reincorporation of exogenous ubiquinone restores at least 90% of these activities. Reduction of the bc1 complex by ETF and acyl CoA oxidase activity are also inhibited by antibody to ETF-QO. Myxothiazole and antimycin which inhibit the quinonol oxidation and quinone reduction sites, respectively, in the bc1 complex also inhibit electron transport from ETF-QO through the complex according to current models of the Q-cycle (Rich, P.R. (1986) J. Bioenerg. Biomembranes 18, 145-156). The results show that ETF-QO is an obligatory component of the electron transport pathway between ETF and the ubiquinone pool and suggest a mechanism for the steady-state turnover of ETF-QO.

Animals↗
PubMed1987

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase.

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.

Antimycin A↗
PubMed1988

Unique action of a modified weakly acidic uncoupler without an acidic group, methylated SF 6847, as an inhibitor of oxidative phosphorylation with no uncoupling activity: possible identity of uncoupler binding protein.

The potent weakly acidic uncoupler SF 6847 was modified by methylation of its phenolic OH group, and the effect of the resulting derivative, with no acid-dissociable group, on oxidative phosphorylation in rat liver mitochondria was examined. The methylated SF 6847 did not induce uncoupling at up to 40 microM, while SF 6847 uncoupled oxidative phosphorylation completely at about 20 nM, indicating that the acid-dissociable group is essential for uncoupling. The O-methylated SF 6847 at 20 microM did, however, inhibit state 3 respiration of mitochondria, although it did not inhibit electron-flow through the respiratory chain, ATPase activated by weakly acidic uncouplers or Pi-ATP exchange. At the same concentration, it also inhibited ATP synthesis in submitochondrial particles. These features are different from those of known inhibitors of oxidative phosphorylation. Thus, O-methylated SF 6847 is a unique inhibitor of oxidative phosphorylation. The possible identity of the uncoupler binding protein is discussed on the basis of these results.

Adenosine Triphosphate↗
PubMed1988

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in isolated rat-liver mitochondria.

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer across the first site of the respiratory chain were performed in isolated rat-liver mitochondria, and the experimental results were compared with the predictions of a simple delocalized chemiosmotic mechanism. The rates of ATP hydrolysis (Jp) and reverse electron transfer (-J0) were measured at different uncoupler (S-13) concentrations, either in the absence or in the presence of rotenone. When the rates -J0 and Jp measured at different uncoupler concentrations were expressed as percentages of the activity at zero uncoupler concentration, it was found that the efficiency of S-13 to uncouple the reverse electron transfer and to stimulate ATP hydrolysis was not significantly changed upon partial inhibition with rotenone. These results are in contrast with data from a study of uncoupler-inhibitor titrations in submitochondrial particles published previously, in which a higher effectiveness of several uncouplers to inhibit ATP-driven reverse electron transfer was observed in the presence of rotenone.

Acetoacetates↗
PubMed1990

Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome.

Liver mitochondria from rats fed ethanol chronically demonstrate an impaired ability to incorporate [35S]methionine into polypeptide products in vitro. This ethanol-induced effect on mitochondrial translation in vitro could not be attributed to significant differences in the methionine precursor pool sizes of ethanol and control mitochondria or to the acute effects of residual ethanol. The observed reduction of radiolabeled methionine incorporation into mitochondrial gene products of ethanol mitochondria in vitro reflects a decrease in the synthesis of all the mitochondrial gene products. However, the percentage of total radiolabel incorporated into each gene product is unaffected by ethanol, suggesting an ethanol-induced coordinate depression of mitochondrial protein synthesis. Moreover, SDS-PAGE and densitometry of submitochondrial particles from ethanol-fed and control rats demonstrated that the steady-state concentration of each of the mitochondrial gene products is decreased in ethanol-fed rats. This reduction of the steady-state concentration of the mitochondrial gene products may be related to the observed depressions of oxidative phosphorylation activities associated with hepatic mitochondria from ethanol-fed rats.

Alcoholism↗
PubMed1990

Nitric oxide formed by nitrite reductase of Paracoccus denitrificans is sufficiently stable to inhibit cytochrome oxidase activity and is reduced by its reductase under aerobic conditions.

Nitric oxide, generated by the action of purified nitrite reductase, inhibited the oxidase activity of both membrane vesicles from anaerobically grown Paracoccus denitrificans and bovine heart submitochondrial particles. In the former case, the inhibition was relatively short-lived and its duration was reduced either by decreasing the concentration of nitrite or raising the ratio of vesicles to nitrite reductase enzyme. These observations indicate that nitric oxide, at least at low concentrations, was sufficiently stable in the presence of oxygen to allow diffusion between proteins in aqueous solution. The shorter inhibition period with P. denitrificans membrane vesicles implies that the nitric oxide reductase of the vesicles is active in the presence of oxygen and has a sufficiently high affinity for nitric oxide to remove it from oxidase enzymes by competition. These observations are related to previous reports of potent inhibition under certain conditions of oxidase activity of P. denitrificans cells by a molecular species produced from nitrite. The implications of the deduced stability of nitric oxide in aerobic solutions are considered with respect to both the phenomenon of aerobic denitrification and the synthesis of nitric oxide in mammalian cells.

Aerobiosis↗
PubMed1993

The proton pumping activity of H(+)-ATPases: an improved fluorescence assay.

A new method for the estimation of steady-state delta pH, and the rate of acidification, by H(+)-ATPases (and other proton transporters) in inverted membrane vesicles is described. The method is based on a combination of two widely used fluorescent delta pH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine. It is demonstrated that 9-amino-6-chloro-2-methoxyacridine fluorescence quenching, which is very sensitive to small pH gradients, is not sensitive to the magnitude of large pH gradient, while 9-aminoacridine, which does not sense small gradients, is very sensitive to large pH gradients. A proper mixture of the two probes provides a method which is equally sensitive to pH gradients from very small values up to 3.5 pH units. The probe response was evaluated by titrations of the fluorescence signal with nigericin and adjusted by changing the concentration ratio and the emission wavelength. In liposomes, submitochondrial particles and bacterial vesicles an almost linear dependence of quenching on delta pH over the entire range can be obtained with this method. It is demonstrated that the new method can be used to obtain more reliable estimates of the rate of acidification as well as the magnitude of delta pH, whereas each of these and similar probes, by themselves are not as reliable. A determination of the ratio delta Gp/delta muH over a wide range of values reveal that this ratio is not constant but decreases with delta Gp. This finding should be taken into consideration when attempting to estimate the H+/ATP ratio form the measurement of delta Gp/delta muH.

Animals↗
PubMed1995

A model of antimycin A binding based on structure-activity studies of synthetic antimycin A analogues.

The structural factors of antimycin A molecule required for inhibitory action were studied using newly synthesized antimycin A derivatives with bovine heart submitochondrial particles, in order to probe the interaction between antimycin A and its binding site. In particular, we focused upon the roles of the amide bond bridge, which connects the salicylic acid and dilactone ring moieties, and the 3-formylamino group in the salicylic acid moiety. The lack of formation of an intramolecular hydrogen-bond between phenolic OH and amide carbonyl groups resulted in a remarkable loss of the activity (by four orders of magnitude), indicating that this hydrogen-bond is essential for the inhibition. This result suggested that both the phenolic OH and the carbonyl groups form a hydrogen-bond with some residues at a fixed conformation. In addition, the inhibitory potency was remarkably decreased by N-methylation of the amide bond moiety, indicating that the NH group might function in hydrogen-bond interaction with the binding site. The N-methylation of 3-formylamino group also resulted in a decrease in the activity, probably due to a loss of the rotational freedom of this functional group. Molecular orbital calculation studies with respect to the conformation of the 3-formylamino group indicated that this group takes an active conformation when the formyl carbonyl projects to the opposite side of the phenolic OH group. Based upon a series of structure-activity studies of synthetic antimycin A analogues, we propose a tentative model for antimycin A binding in its binding cavity.

Amides↗