valinomycin and malic-acid

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

Topics: Animals; Brain; Glutamic Acid; Hydrogen Peroxide; Hydrogen-Ion Concentration; Malates; Membrane Potentials; Mitochondria; Models, Biological; Models, Chemical; Rats; Reactive Oxygen Species; Rotenone; Spectrometry, Fluorescence; Valinomycin

Inside-out subcellular vesicles of Azotobacter vinelandii are found to produce delta pH and delta psi (interior acidic and positive) when oxidising malate or menadiol. These effects are inherent in both Cyd+ Cyo- (lacking the o-type oxidase) and Cyd- Cyo+ (lacking the bd-type oxidase) strains. They appear to be myxothiazol-sensitive in the Cyd- Cyo+ strain but not in the Cyd+ Cyo- strain. The H+/e- ratio for the terminal part of respiratory chain of a bd-type oxidase overproducing strain is established as being close to 1. It is also shown that NADH oxidation by the vesicles from the Cyd- Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+ Cyo- strain is resistant to these Q-cycle inhibitors. It is concluded that (i) the bd-type oxidase of A. vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase and (ii) Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A. vinelandii respiratory chain and does not in the bd-type quinol oxidase terminated branch. These relationships are discussed in the context of the respiratory protection function of the bd-type oxidase in A. vinelandii.

Topics: Anaerobiosis; Azotobacter vinelandii; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Cytochrome b Group; Cytochromes; Electron Transport Chain Complex Proteins; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Malates; Methacrylates; Oxidoreductases; Subcellular Fractions; Thiazoles; Valinomycin; Vitamin K

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.

Topics: Ammonia; Animals; Aspartic Acid; Cytoplasm; Energy Metabolism; Gluconeogenesis; Lactates; Lactic Acid; Liver; Malates; Male; Mitochondria, Liver; NAD; Oxidation-Reduction; Palmitates; Rats; Rats, Inbred Strains; Valinomycin

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.

Topics: Animals; Biological Transport; Coenzyme A; Electrochemistry; Hydrogen-Ion Concentration; Intracellular Membranes; Ketoglutaric Acids; Malates; Male; Mitochondria, Heart; Nigericin; Phosphates; Protons; Rats; Rats, Inbred Strains; Succinates; Succinic Acid; Valinomycin

The limiting factors of the involvement of malate dehydrogenase in mitochondrial malate oxidation were investigated by using Percoll-purified potato tuber mitochondria. The respective roles of reduced pyridine nucleotides, oxaloacetate, and adenine nucleotides were studied under conditions of high or low phosphorylation potential (Pi + ADP/ATP ratio). Under conditions of high phosphorylation potential, the limitation of malate dehydrogenase activity was caused by the accumulation of oxaloacetate in the medium. In the absence of ADP (phosphorylation potential close to zero), ATP was responsible for the inhibition of malate dehydrogenase activity rather than oxaloacetate or reduced pyridine nucleotides.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Biological Transport; Enzyme Activation; Malate Dehydrogenase; Malates; Mitochondria; Oxidation-Reduction; Plants; Potassium; Valinomycin

ATP synthesis by starved whole cells of alkalophilic Bacillus firmus RAB was energized by addition of DL-malate or by imposition of a valinomycin-mediated K+ diffusion potential. At pH 9.0, the transmembrane electrical potentials produced by these two means were similar in magnitude, at close to -170 mV. While N,N'-dicyclohexylcarbodiimide-sensitive ATP synthesis occurred upon the addition of DL-malate, no ATP was synthesized in response to a diffusion potential. In contrast, Na+-dependent accumulation of alpha-aminoisobutyric acid was energized equally well by DL-malate or a diffusion potential at pH 9.0. Even at pH 7.0, DL-malate was more efficacious than a diffusion potential in energizing ATP synthesis as assessed by determining the phosphorylation potentials generated at transmembrane electrical potential values of different magnitudes. Both modes of energization did, however, result in N,N'-dicyclohexylcarbodiimide-sensitive ATP synthesis at pH 7.0. The results of these studies are consistent with a model of energy coupling in which one pathway of protons between the respiratory proton pumps and the H+-translocating ATPase is direct or localized. Whereas an artificially imposed electrochemical gradient of protons can energize ATP synthesis under certain experimental conditions, in the proton-poor milieu that is optimal for growth of alkalophilic bacilli, the direct proton pathway may be crucial.

Topics: Adenosine Triphosphate; Bacillus; Cell Membrane; Dicyclohexylcarbodiimide; Diffusion; Hydrogen-Ion Concentration; Malates; Membrane Potentials; Oxygen Consumption; Thermodynamics; Valinomycin