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The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange.

ABSTRACT: 1. Increasing the substrate concentration only decreased the inhibition of mitochondrial oxidations by diphenyleneiodonium or by 2,4-dichlorophenyleneiodonium by a small amount. 2. Diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium lowered the amounts of succinate, citrate and glutamate accumulated in the matrix of mitochondria in the presence of Cl-, but not in its absences. 2,4-Dichlorodiphenyleneiodonium decreased the accumulation of substrates by mitochondria oxidizing glycerol 3-phosphate. 3. Diphenyleneiodonium caused an alkalinization of the medium with an anaerobic suspension of mitochondria, which was only partly reversed by Triton X-100. 4. The rate of proton extrusion by mitochondria oxidizing succinate was not altered by diphenyleneiodonium or by 2,4-dichlorodiphenyleneiodium, although the rate of decay of proton pulses was increased. 5. 2,4-Dichlorodiphenyleneiodonium shifted the pH optimum for succinate oxidation by intact mitochondria from pH 7.2 to 8.0, whereas there was no effect on that of freeze-thawed mitochondria, which was pH 8.0. 6. The concentration of 2,4-dichlorophenyleneiodonium required to inhibit respiration by 50% is less the higher the absolute rate of oxygen uptake. 7. EDTA, but not EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid] increased the inhibition of respiration by diphenyleneiodonium, 2,4-dichlorodiphenyleneiodonium and by tri-n-propyltin. 8. It is concluded that diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium limit respiration in Cl--containing medium by causing an acidification of the matrix, and that there are pH-sensitive sites in the respiratory chain between NADH and succinate, and between succinate and cytochrome c.

SUBMITTER: Gatley SJ

PROVIDER: S-EPMC1163973 | biostudies-other | 1976 Aug

REPOSITORIES: biostudies-other

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Project description:1. The hypoglycaemic compound diphenyleneiodonium causes rapid and extensive swelling of rat liver mitochondria suspended in 150mm-NH(4)Cl, and in 150mm-KCl in the presence of 2,4-dinitrophenol and valinomycin. This indicates that diphenyleneiodonium catalyses a compulsory exchange of OH(-) for Cl(-) across the mitochondrial inner membrane. Br(-) and SCN(-) were the only other anions found whose exchange for OH(-) is catalysed by diphenyleneiodonium. 2. Diphenyleneiodonium inhibited state 3 respiration of mitochondria and slightly stimulated state 4 respiration with succinate or glutamate as substrate in a standard Cl(-)-containing medium. 3. Diphenyleneiodonium did not inhibit state 3 respiration significantly in two Cl(-)-free media (based on glycerol 2-phosphate or sucrose) but caused some stimulation of state 4. 4. In Cl(-)-containing medium diphenyleneiodonium only slightly inhibited the 2,4-dinitrophenol-stimulated adenosine triphosphatase and it had little effect in the absence of Cl(-). 5. The inhibition of respiration in the presence of Cl(-) is dependent on the Cl(-)-OH(-) exchange. 2,4-Dichlorodiphenyleneiodonium is ten times as active as diphenyleneiodonium both in causing swelling of mitochondria suspended in 150mm-NH(4)Cl and in inhibiting state 3 respiration in Cl(-)-containing medium. Indirect evidence suggests that the Cl(-)-OH(-) exchange impairs the rate of uptake of substrate anions. 6. It is proposed that stimulation of state 4 respiration in the absence of Cl(-) depends, at least in part, on an electrogenic uptake of diphenyleneiodonium cations. 7. Tripropyl-lead acetate, methylmercuric iodide and nine substituted diphenyleneiodonium derivatives also catalyse Cl(-)-OH(-) exchange across the mitochondrial membrane. 8. Diphenyleneiodonium is compared with the trialkyltin compounds, which are also known to mediate Cl(-)-OH(-) exchange and which have in addition strong oligomycin-like effects on respiration. It is concluded that diphenyleneiodonium is specific for catalysing anion-OH(-) exchange and will be a useful reagent for investigating membrane-dependent systems.

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The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange.

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