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PubMed1997

Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.

Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.

Adenosine Triphosphatases↗
PubMed1997

4,6-Dinitro-o-cresol uncouples oxidative phosphorylation and induces membrane permeability transition in rat liver mitochondria.

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.

2,4-Dinitrophenol↗
PubMed1999

Stereoselective and substrate-dependent inhibition of hepatic mitochondria beta-oxidation and oxidative phosphorylation by the non-steroidal anti-inflammatory drugs ibuprofen, flurbiprofen, and ketorolac.

Non-steroidal anti-inflammatory drugs (NSAIDs) cause a range of adverse effects, some of which have been associated with perturbances of lipid metabolic pathways. Previous data demonstrating stereoselective formation of the CoA thioester of R-ibuprofen in particular were suggestive of possible stereoselective effects on lipid metabolism. Our aim was to characterise the relative stereoselectivity of the effects of ibuprofen, flurbiprofen, and ketorolac (0.01-1.0 mM) on both the beta-oxidation of palmitate and oxidative phosphorylation in rat hepatic mitochondria as a means of dissecting prostaglandin related from non-prostaglandin-related events. Beta-oxidation was inhibited stereoselectively by R-ibuprofen (P = 0.015), non-stereoselectively by R- and S-flurbiprofen (P = 0.002 and P = 0.004, respectively), and was essentially unaffected by either enantiomer of ketorolac. At 0.25 mM, inhibition by R-ibuprofen and both flurbiprofen enantiomers was partially reversed by increasing CoA concentrations (0-200 microM). Mitochondrial respiration was moderately inhibited by both enantiomers of ibuprofen and flurbiprofen (P < 0.01), but only by high concentrations (> or = 1 mM) of the enantiomers of ketorolac (P < 0.01). Uncoupling of oxidative phosphorylation measured as stimulation of State 4 respiration contributed to these effects. The data support interactions involving both stereoselective CoA-dependent and non-CoA-dependent mechanisms. The plasma drug concentrations required to achieve these effects are not likely to be attained in the majority of patients, although these concentrations are achievable in the gastrointestinal tract and may contribute to the well-known spectrum of adverse effects in this organ. Some patients do experience systemic adverse events which may be mediated by these mechanisms.

Animals↗
PubMed1975

Ethidium bromide inhibits mitochondrial phosphorylating oxidation.

Ethidium bromide, in addition to combination with mitochondrial nucleic acids, is a phosphorylation inhibitor during glutamate and succinate respiration by mitochondria. Exhaustive washing of ethidium bromide-treated mitochondria did not relieve the inhibition nor significantly decrease the amount of bound dye. Dialysis against a cation exchange resin at 3 degrees for 17 hr removed about 97% of bound dye. This restored phosphorylating capacity to that of untreated mitochondria which had also been dialyzed against the resin. Since state 3 respiration was diminished and state 4 was unaffected by the presence of the acridine dye, and since neither swelling of mitochondria nor release of latent ATPase was observed, then ethidium bromide was not an electron transport inhibitor nor an uncoupler of oxidative phosphorylation. Inhibition of metabolic processes by ethidium bromide may be due in part to depressed generation of mitochondrial ATP.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Effect of protein-markers of growth on oxidative phosphorylation in mitochondria].

Growth marker proteins (GMP) were studied for their effect on oxidative phosphorylation in the heart and liver mitochondria of rabbits. It is shown that GMP decrease a respiratory control (RC) coefficient, P/O coefficient, inhibit respiration of the mitochondria in metabolic states 3, 5 and activates it in state 4. The nature of the oxidation substrates (FAD- and NAD-dependent succinic and pyruvic acids, respectively) does not influence the GMP effect manifestation. It is supposed that GMP disturb the structural and functional integrity of the mitochondria. Variations in bioenergetic parameters of the heart and liver mitochondria in organisms with active growth foci as well as of mitochondria incubated with GMP, are unidirectional. Cytochrome c, coenzyme A (Co ASH) and other thyol compounds (cystein, dithiotreitol, glutathione--GSH) remove the GMP action.

Animals↗
PubMed1980

Anisotropic inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by tetraphenylarsonium.

Tetraphenylarsonium (TPA+) inhibited energy transduction in oxidative phosphorylation in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. TPA+ incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. TPA+ also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced binding sites on the membranes for TPA+. The amounts of energy-dependent binding sites for TPA+ on mitochondria energized with succinate and with ATP, respectively, were 90 and 13 nmol/mg of protein. TPA+ also caused shrinkage of mitochondria energized with succinate and with ATP in an energy-dependent fashion. The energy-dependent binding of TPA+, TPA+-induced H+-ejection, TPA+-induced shrinkage of mitochondria, and TPA+-induced inhibition of energy transduction occurred in parallel. The present findings show that TPA+ inhibits energy transduction by binding to negative charges created on lipophilic domains near the surface of the outer side (C-side) of the mitochondrial inner membranes, and that it has no inhibitory activity on the inner side (M-side) of the membranes.

Adenosine Triphosphate↗
PubMed2004

Congenital hypertrophic cardiomyopathy, cataract, mitochondrial myopathy and defective oxidative phosphorylation in two siblings with Sengers-like syndrome.

UNLABELLED: We describe two siblings with a Sengers-like syndrome, who presented with congenital hypertrophic cardiomyopathy, infantile cataract, mitochondrial myopathy, lactic acidosis and normal mental development. A mitochondrial adenine nucleotide translocator 1 (ANT1) defect was detected since the ANT1 protein was not detectable by immmunoblotting in muscle samples of the patients. Additionally to these features of classical Sengers syndrome (OMIM 212350), we found that the mitochondrial oxidative phosphorylation, measured by biochemical analysis, was severely compromised in skeletal muscle in both children. Biochemical and morphological analysis of the fibroblasts revealed normal results. The association of significantly decreased pyruvate oxidation rates, deficient energy production and decreased multiple mitochondrial enzyme-complex activities in the muscle samples of our patients is a new finding which differs from previous results in patients with Sengers syndrome. CONCLUSION: we recommend a muscle biopsy and the biochemical analysis of the oxidative phosphorylation system in patients with muscle hypotonia, cardiomyopathy and congenital or infantile cataract.

Acidosis, Lactic↗
PubMed1986

Photophosphorylation and oxidative phosphorylation in intact cells and chromatophores of an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Light-induced ATP synthesis was studied in intact cells and chromatophores of Erythrobacter sp. strain OCh114. ATP synthesis was measured by both the pH method and the luciferin-luciferase luminescence method. The rate of ATP synthesis was moderate (a typical value of 0.65 mol of ATP per mol of bacteriochlorophyll per min), and synthesis was inhibited by antimycin A. ATP was synthesized under illumination only under aerobic conditions and not under anaerobic conditions. This characteristic was similar to that of other light-induced energy transduction processes in this bacterial species, such as oxidation of reaction center, oxidation of cytochrome c551, and translocation of H+, which were not observed under anaerobic conditions. This phenomenon was reconciled with the fact that the Erythrobacter sp. could not grow anaerobically even in the light. The characteristics of oxidative phosphorylation and ATP hydrolysis were also investigated. The respiratory ratio of chromatophores was 2.3. Typical rates of oxidative phosphorylation by NADH and by succinate were 2.9 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.22) and 1.1 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.19), respectively. A typical rate of ATP hydrolysis was 0.25 mol of ATP per mol of bacteriochlorophyll per min in chromatophores. ATPase and adenylate kinase are also involved in the metabolism of adenine nucleotides in this bacterium.

Adenosine Triphosphate↗
PubMedDate not supplied

[Effect of sodium hydroxybutyrate on mitochondrial oxidative phosphorylation and myocardial pentose cycle enzymes during high altitude hypoxia].

The effect of intraperitoneal administration of sodium hydroxybutyrate on oxidative phosphorylation in mitochondria and on the activity of glucoso-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cardiac muscle was studied under alpine conditions (3200 m above the sea level). The drug was shown to increase the conjugation of oxidation with phosphorylation, to potentiate the activity of the test enzymes of the pentose cycle in the heart of the animals with one-month long adaptation to high-altitude hypoxia.

Acclimatization↗
PubMedDate not supplied

[Effect of insulin on oxidative phosphorylation in the liver and heart mitochondria of adrenalectomized rats].

The effect of insulin was studied as applied to the inhibited under conditions of adrenalectomy process of oxidative phosphorylation in the rat liver and heart mitochondria. It is established that adrenalectomy does not change oxidative activity of mitochondria but inhibits the process of phosphorylation, which results in the decreased values of the ADP/O coefficient and the respiratory control. Insulin administered to the adrenalectomized rats 3h before the experiments reduces the disturbed oxidative phosphorylation in mitochondria of the liver and heart by intensifying the degree of ADP phosphorylation.

Adenosine Diphosphate↗
PubMed1997

Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases.

Inhibitor titrations were applied to characterize functional changes in mitochondrial energy metabolism in the skeletal muscle of patients with mitochondrial diseases. For this we titrated the maximal mitochondrial respiration rate of saponin-skinned muscle fibers isolated from the skeletal muscle biopsy with the specific inhibitors of mitochondrial oxidative phosphorylation complexes I, IV and V-rotenone, azide and oligomycin. For three patients with deletions of mitochondrial DNA and one patient with a complex I deficiency the titrations revealed at rather normal respiration activities of saponin-skinned fibers significant differences to healthy controls: (i) The inhibitor titration curves of the affected enzyme were much steeper and (ii) for almost complete inhibition of respiration a smaller amount of the inhibitor is necessary. The detailed analysis of the titration curves within the framework of metabolic control theory indicated elevated flux control coefficients of the respective complex of respiratory chain. On the other hand, for one patient with a mitochondrial DNA depletion syndrome, decreased respiration activities of skinned fibers but no redistribution of flux control was observed. We conclude, therefore, that application of inhibitor titrations and the quantitative description of the titration curve can be a valuable approach to elucidate functional defects of mitochondrial oxidative phosphorylation.

Adult↗
PubMed1995

Cellular targets of 3'-azido-3'-deoxythymidine: an early (non-delayed) effect on oxidative phosphorylation.

Previous results demonstrated that incubation of the Friend murine erythroleukemic cell with 5 microM AZT for several days leads to a decrease in the rate of cell growth, inhibition of mtDNA replication, reduction of mtDNA per cell and per mitochondrion, and an increase in mitochondria per cell. As shown here, such treatment also leads to changes in lactate and ATP synthesis and in O2 uptake, suggesting impairment of oxidative phosphorylation. Direct measurement of ATP synthesis in mitochondria isolated from AZT-treated cells confirmed this view. The most significant new finding in this paper, however, is that in addition to these delayed effects of AZT, similar but very rapidly appearing effects on oxidative phosphorylation were noted, with changes observed in the above parameters including mitochondrial proliferation. Some of these occurred as early as 3 hr, only 7% of the doubling time, after exposure of the cells to 5 microM AZT, a period too short for initiation of appreciable mtDNA-mediated effects. Studies on isolated mitochondria provided no evidence of the identity of the immediate target of AZT: AZT does not act as an uncoupler or inhibitor of respiratory control, and previous results failed to implicate adenylate kinase. We have also begun to address the question of the mechanism of AZT-induced mitochondrial proliferation. Initial experiments showed that AZT inhibited synthesis of total cytosolic protein but stimulated synthesis of those proteins imported into mitochondria from the cytoplasm. We also report that aminothymidine, a catabolite of AZT in liver capable of inhibiting cell growth, was not generated by Friend cells.

Adenosine Triphosphate↗
PubMed1979

Steady-state coupling of four membrane systems in mitochondrial oxidative phosphorylation.

According to Alexandre, Reynafarje, and Lehninger, four different membrane systems are involved, with definite stoichiometry, in the mitochondrial synthesis of ATP by electron transport, via proton transport. We adopt this model and pursue some of its thermodynamic consequences. At steady state, each of the four systems must have the same flux J through the membrane and the overall thermodynamic force X for oxidative phosphorylation is the sum of the four separate forces. From these properties, using an empirical linear flux-force relation for each system, it is easy to obtain J as a function of X. In turn, X depends on the inside [NAD+]/[NADH] and the outside [ATP]/[ADP][Pi] quotients (and on the pH inside). Thus, J is related to these quotients. The relationship we derive is similar to that described by Erecińska and Wilson, as deduced from a quite different model of oxidative phosphorylation. Proton transport is involved explicitly in three of the four systems of the present model. However, because of the steady-state stoichiometric coupling of the four systems, proton transport does not appear in the overall reaction. On the other hand, Erecińska and Wilson use, in their model, a direct connection between electron transport and ATP synthesis. The present paper demonstrates that J can be related to the quotients mentioned above without this direct connection.

Intracellular Membranes↗
PubMed2004

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation.

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.

Animals↗
PubMed1987

4-Chloro-4'-biphenylol as an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation.

4-Chloro-4'-biphenylol (4'-OH-4-CB), a metabolite of 4-chlorobiphenyl (4-CB), stimulated state 4 respiration and released oligomycin-inhibited state 3 respiration of rat liver mitochondria with succinate as the respiratory substrate. When glutamate/malate and beta-hydroxybutyrate were used as the substrates, however, 4'-OH-4-CB was ineffective on these parameters. This indicates that 4'-OH-4-CB uncouples oxidative phosphorylation with succinate, but not with glutamate/malate and beta-hydroxybutyrate. 4'-OH-4-CB severely inhibited 2,4-dinitrophenol (DNP)-stimulated respiration with glutamate/malate (ID50, 25 microM) and beta-hydroxybutyrate (ID50, 32 microM) because of the blockade of electron transfer between NADH and CoQ span, masking the uncoupling action of 4'-OH-4-CB. On the other hand, the inhibition of the respiration with succinate was only apparent at high 4'-OH-4-CB concentrations (ID50, 260 microM). 4'-OH-4-CB also inhibited the oxidation of NADH in submitochondrial particles (ID50, 35 microM). State 3 respiration was more intensely inhibited by 4'-OH-4-CB in the presence of either glutamate/malate (ID50, 23 microM) or beta-hydroxybutyrate (ID50, 26 microM) than that in the presence of succinate (ID50, 220 microM). Thus, 4'-OH-4-CB acts as both an uncoupler and an inhibitor of oxidative phosphorylation. The overall in vitro effect is to prevent ATP synthesis, which may be an important factor in the mechanism underlying the toxicity of 4-CB.

Adenosine Triphosphate↗
PubMed1991

On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare).

Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.

Journal Article↗
PubMed1995

Mitochondria: beyond oxidative phosphorylation.

Mitochondria are essential eukaryotic organelles that perform many functions in addition to oxidative phosphorylation. Some of these functions are still poorly understood or as yet undiscovered, but may resemble already known functions in bacteria. The rapidly growing sequence information on bacterial genes, the polymerase chain reaction, and gene disruption in yeast thus offer a powerful approach for discovering new mitochondrial functions.

ATP-Dependent Proteases↗