Absence Of Oligomycin Research Articles

Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.

Release of mitochondrial respiratory control by cyanate salts

Mitochondrial respiration is stimulated by 5–40 mM potassium cyanate in the presence or absence of oligomycin. When the cyanate concentration is increased over 40 mM, the mitochondria respire at progressively lower rates. In these respects, although at relatively high concentrations, cyanate behaves as an uncoupler of oxidative phosphorylation.

Sensitivity of the (Na + + K +)-ATPase to state-dependent inhibitors : Effects of digitonin and triton X-100

Treatment of a purified (Na + + K +)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na + + K +)-ATPase reaction inhibited more than the K +-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na + + K +)-ATPase reaction but not the K +-phosphatase reaction; however, treatment with digitonin made the K +-phosphatase reaction almost as sensitive to oligomycin as the (Na + + K +)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K +-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K +-phosphatase reaction more than the (Na + + K +)-ATPase reaction). Both digitonin and Triton markedly increased the K 0.5 for K + as activator of the K +-phosphatase reaction, with little effect on the K 0.5 for K + as activator of the (Na + + K +)-ATPase reaction. In contrast, increasing the K 0.5 for K + in the K +-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K +-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E 1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E 2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K 0.5 for K + in the K +-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na + plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na + plus nucleotide.

Oligomycin-resistant mitochondrial ATPase from mouse fibroblasts

Fourteen oligomycin-resistant LM(TK-) clones were isolated following the mutagenesis of minicells. In the absence of oligomycin, the mutants grew with population doubling times similar to that of the wild type (1 day). In 3 or 5 microgram oligomycin/ml the doubling times of the mutants were 1.2-2.5 days. Both stable and unstable classes were represented among the oligomycin-resistant mutants. Mitochondrial ATPase activities of the mutants were 1.3-1130 times more resistant to oligomycin than the wild type. The mitochondrial ATPase of OLI 14 was found to be bound firmly to the mitochondrial membrane, showed no alteration in the pH optimum compared to wild-type, and exhibited increased resistance to DCCD and venturicidin. These results are consistent with the conclusion that oligomycin resistance in these mutants results from altered mitochondrial ATPase.

Identification of an intramitochondrially synthesized proteolipid associated with the mitochondrial ATPase complex as the product of a mitochondrial gene determining oligomycin resistance in Aspergillus nidulans.

An extranuclear oligomycin-resistant mutant of Aspergillus nidulans was isolated and biochemically analyzed. The mutant grows slowly in the absence of oligomycin and contains an excess of cytochrome c. The ATPase complex solubilized from mutant mitochondria with Triton X-100 is 80 times more resistant to oligomycin than the enzyme of the parental strain. The enzyme complexes of both strains were further purified to electrophoretic homogeneity and resolved by dodecylsulfate gel electrophoresis in the presence of 8 M urea into 16 polypeptide bands of apparent molecular weights between 65000 and 6000. Four proteins of apparent molecular weights 43000, 21000, 11000 and 6000 are synthesized on cycloheximide-resistant mitochondrial ribosomes. The smallest subunit is selectively extracted from the purified enzyme by neutral chloroform/methanol (2/1). A proteolipid of identical electrophoretic mobility and site of synthesis is the major component of proteins extractable with the same solvent from whole mitochondrial membranes. This protein has been isolated from the mutant and its parental strain, and purified to apparent homogeneity. The proteins from both strains start with N-formyl-methionine and end with valine. However, a second internal methionine residue present in the wild-type protein is absent, together with some other amino acids, from the mutant protein. It is concluded that this small lipophilic protein is coded by a mitochondrial gene determining oligomycin resistance, is synthesized on mitochondrial ribosomes and is associated with the membrane sector of the ATPase complex. The mutational alteration of the protein not only confers oligomycin resistance to the ATPase complex but also causes a structural alteration of the mitochondrial inner membrane.

Regulation of Glutamate Metabolism and Interactions with the Citric Acid Cycle in Rat Heart Mitochondria

Abstract The interactions of glutamate metabolism with transport steps across the mitochondrial membrane and enzyme steps of the citric acid cycle have been investigated in rat heart mitochondria in order to elucidate factors regulating transfer of reducing equivalents into mitochondria via the malateaspartate cycle. With glutamate as substrate, net efflux of α-ketoglutarate from the mitochondria in State 3 was stimulated by addition of extramitochondrial malate, the concentration for a half-maximal effect being 0.36 ± 0.07 mm. Stimulation of α-ketoglutarate-malate exchange by a decrease of the malate gradient resulted in a fall of the intramitochondrial α-ketoglutarate concentration and diminished flux through α-ketoglutarate dehydrogenase. A value of 0.67 mm for the apparent Km of α-ketoglutarate dehydrogenase for α-ketoglutarate was calculated from these data. Glutamate transamination was inhibited in the uncoupled state and the intramitochondrial aspartate content increased up to 20-fold. The increased aspartate gradient across the mitochondrial membrane observed in the energy-depleted state was abolished, while aspartate efflux and glutamate transamination were restored either by direct addition of ATP or by addition of acetylcarnitine to increase substrate level phosphorylation. These effects were prevented by oligomycin, indicating that activation of aspartate efflux required energy in a form closely associated with the energy transfer reactions of oxidative phosphorylation. Regulation of flux through citrate synthase, aspartate aminotransferase, and α-ketoglutarate dehydrogenase as a function of the state of phosphorylation of the mitochondrial adenine nucleotides was investigated at high NADH:NAD ratios produced by State 4 and oligomycin inhibition, and at low NADH:NAD ratios produced by addition of uncoupling agents in the presence and absence of oligomycin. Acetylcarnitine and acetoacetate were used to provide sources of intramitochondrial acetyl coenzyme A. The following conclusions may be drawn from the data. In addition to the malate concentration gradient, net α-ketoglutarate efflux is regulated by the rate of generation of intramitochondrial α-ketoglutarate relative to the activity of α-ketoglutarate dehydrogenase as determined by product inhibition. Flux through α-ketoglutarate dehydrogenase was inhibited at high ATP:ADP ratios due to the high succinyl-CoA to CoA ratio, with the inhibition potentiated by an increase of the NADH:NAD ratio and relieved by an increased availability of intramitochondrial α-ketoglutarate. Production of α-ketoglutarate via citrate synthase was also regulated by the phosphorylation state of the intramitochondrial adenine nucleotides as indicated by succinyl-CoA inhibition rather than by direct ATP inhibition. Since inhibition of citrate synthase by succinyl-CoA has been shown to be competitive with acetyl-CoA, it is proposed that regulation is exerted by changes of the succinyl-CoA to acetyl-CoA ratio. This regulation is complementary to control of oxalacetate availability by the intramitochondrial NADH:NAD ratio which also regulates α-ketoglutarate production via both citrate synthase and aspartate aminotransferase. Coordination of flux between enzymes which produce intramitochondrial α-ketoglutarate, namely citrate synthase and aspartate aminotransferase, and α-ketoglutarate dehydrogenase which utilizes it, is determined by complex feedback relationships regulated indirectly both by the ATP:ADP ratio and the NADH:NAD ratio. These provide great flexibility to regulation of the over-all malate-aspartate cycle by controlling efflux of α-ketoglutarate from the mitochondria.

Respiration-driven Proton Transport in Submitochondrial Particles

Abstract 1. The maximal H+:O ratios of proton transport in highly resolved submitochondrial particles prepared by sonication of bovine heart mitochondria in the presence of ammonia and exposed sequentially to Sephadex and urea (ASU particles) in the presence of oligomycin were found to be 6, 4, and 2 during the oxidation of NADH, succinate, or N-methylphenazonium methosulfate, respectively. A pH dependence for the H+:O ratio was observed with optimal ratios at pH 6.0 to 6.5. 2. Comparison of the effects of an uncoupler (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and of oligo-mycin on proton transport and on respiratory control in ASU particles showed that the effect of these compounds on the respiratory rate could be accounted for by their effect on the proton permeability of the particles. 3. The external and internal buffering powers of ASU particles were calculated from the extent of overshoot of pH change on addition of acid or alkali. 4. Coupling factors of oxidative phosphorylation, F1 and oligomycin sensitivity-conferring protein, decreased the proton permeability of ASU particles and allowed proton transport to be observed in the absence of oligomycin.

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV max,K i andK m values. The normalV max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV max was accompanied by a time-dependent decrease in (K i)ADP, but there was no change in (K m)ATP. The increase inV max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV max was increased by the activating anions by the same factor as theV max in absence of oligomycin. Certain anions, notably azide, decreasedV max, but did not affect (K i)ADP or (K m)ATP. The decrease inV max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria. The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V max)ATP compared with (V max)GTP. The values of (K m)ATP and (K m)GTP were virtually the same. Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.

The “energized state” of mitochondria: Lifetime and ATP equivalence

1. 1. Aequorin, a bioluminescent jellyfish protein, can measure the concentration of free Ca 2+ in a rapid, sensitive and specific manner, in biological systems. 2. 2. The movements of the nanomolar concentration of mitochondrial “endogenous” Ca 2+ have been studied, and found to be a reversible indicator of the energization of mitochondria. 3. 3. The comparison between the kinetics of cytochromes, changes on addition of respiratory inhibitors, and of the release of endogenous mitochondrial Ca 2+ has brought to the conclusion that the lifetime of the energized states is 7 ± 2 sec in rat-liver mitochondria at 25°. 4. 4. The energy content of the energized state was also determined, by comparing the kinetics of Ca 2+ efflux from mitochondria in the presence and absence of oligomycin and measuring the content of endogenous ATP. It was found to be 0.3 μmole ATP equivalents per g mitochondrial protein. 5. 5. The endogenous mitochondrial Ca 2+ has been found to be in a highly dynamic state. Therefore the proton movements observed on activation of respiration can be considered not primary, but associated with ion movements.

The influence of oligomycin on the actions of epinephrine and theophylline upon the perfused rat heart

Rat hearts were perfused with media containing pyruvate and iodoacetate with and without oligomycin. In the absence of oligomycin, both heart rate and force of contraction were well maintained. When oligomycin was included in the perfusion medium, there was a negative inotropic effect, a marked atrioventricular (A-V) block and a slowly developing contracture. In the oligomycin-treated heart, epinephrine or theophylline increased pacemaker activity, reduced the extent of A-V blockade and greatly accelerated the development of contracture. Rapid occurrence of contracture also followed electrical stimulation. Epinephrine was capable of exerting a positive inotropic effect in electrically driven hearts perfused with iodoacetate, pyruvate and oligomycin. Measurements of adenine nucleotides and creatine-phosphate showed that at the time when heart rate was markedly depressed by oligomycin ATP concentrations were not greatly reduced, although creatine-phosphate levels had decreased by 40 per cent. Administration of epinephrine or theophylline to these hearts greatly diminished the levels of high-energy phosphate. It was concluded that formation of new ATP from glycolysis or the respiratory chain is not required for the initial inotropic action of epinephrine or theophylline. The data allow one to speculate that the primary effects of these compounds or of 3′,5′ cyclic AMP may be concerned with calcium movements in the myocardial cell.

Respiration in Isolated Fat Cells and the Effects of Epinephrine

1. A technique was devised for ready sequential sampling of lipocyte suspensions for measurement of metabolic activities. 2. With the use of this technique in paired incubations, an effect of oligomycin to decrease the QO2 of fat cells to 45% of control was shown. 3. An analogy between the effect of oligomycin on intact cells and that described for isolated mitochondria was suggested, since the antibiotic inhibited incorporation of 32Pi into acid-soluble mononucleotides and decreased 14CO2 production from glucose-1-14C and glucose-6-14C. 4. Stimulation of QO2 by epinephrine was shown in the presence and in the absence of oligomycin; there was no accompanying increase in 14CO2 production from glucose when oligomycin was present, indicating that glucose was not the substrate for the observed increase in oxygen consumption. 5. It is concluded that oligomycin inhibits oxidative phosphorylation in the fat cell. Under these conditions, (a) a significant portion of the respiration of the lipocyte is not linked in an obligatory fashion to phosphorylation, and (b) it may be inferred that the calorigenic effect of epinephrine in adipose tissue involves electron flow to oxygen not linked to phosphorylation.

Succinate-linked nicotinamide-adenine dinucleotide reduction coupled with the aerobic oxidation of reduced tetramethyl-p-phenylenediamine in submitochondrial particles

The energy-dependent reduction of NAD+ by succinate catalyzed by submitochondrial particles derived from beef heart is demonstrated to be supported energetically not only by the addition of ATP but also by a high-energy intermediate generated by the flow of electrons through the cytochrome oxidase portion of the respiratory chain. As substrate for the energy generation is used ascorbate and tetramethyl-p-phenylenediamine. Phenazinemethosulfate but not cytochrome c can replace the phenylenediamine. The reduction does not require the addition of Mg2+, phosphate or nucleotides, it is not inhibited by EDTA or oligomycin and by arsenate only in the absence of oligomycin, when the generated high-energy intermediate is the energy source. Rotenone is inhibitory whereas antimycin A is an obligatory addition to avoid reoxidation of the formed NADH.

The effect of oligomycin on net movements of sodium and potassium in mammalian cells in vitro

1. 1. The effects of oligomycin on the net accumulation of potassium and net loss of sodium by whole mammalian cells have been studied. Three types of cell system, differing in the relationship of the cation movements to the energy-providing metabolism, were used. 2. 2. In liver slices prepared from adult rats oligomycin inhibited sodium and potassium movements by a maximum of 50%. Half-maximal inhibition of potassium uptake was given by oligomycin at a concentration of approx. 3 μg/ml (0.29 μg/mg liver-slice protein). The 50% of the potassium uptake which persisted in the presence of oligomycin was largely inhibited by the further addition of cyanide or 2,4-dinitrophenol. 3. 3. Oligomycin inhibited respiration of the adult tissue by a maximum of 20%, half-maximal inhibition being given by 3 μg/ml oligomycin (0.29 μg/mg protein). The inhibition was released by the further addition of dinitrophenol. 4. 4. Slices of liver prepared from rat foetuses during the last 2 days of gestation can accumulate a considerable amount of potassium when incubated in the presence of cyanide. Oligomycin gave approx. 50% inhibition of this cyanide-resistant accumulation of potassium, the half-maximal effect being given by an oligomycin concentration of about 3 μg/ml (0.41 μg/mg liver-slice protein). In the absence of oligomycin, dinitrophenol almost completely inhibited cyanide-resistant potassium accumulation. In the presence of oligomycin, part of the potassium accumulation remained uninhibited by dinitrophenol. 5. 5. Oligomycin did not inhibit anaerobic glycolysis in the slices of foetal liver. 6. 6. Oligomycin, at concentrations of 10 and 20 μg/ml, had no effect upon net sodium movements in human erythrocytes. In contrast, strophanthin G inhibited the sodium movements. 7. 7. Consideration of these results in relation to the known effects of oligomycin on enzyme activities in subcellular particles suggests that oligomycin probably inhibited cation movements in liver slices by virtue of its inhibitory effect upon oxidative phosphorylation, rather than by inhibition of a reaction directly involved in cation transport. If this conclusion is correct, it follows that part of the energy required for cation transport by liver cells can be derived directly from an energy-rich intermediate of oxidative phosphorylation.