The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract

The influence of the sulfhydryl reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DNTB) on metabolism of liver mitochondria was studied under different experimental conditions. 1. 1. DTNB prevented the stimulation of respiration produced by ADP and inorganic phosphate in the presence of glutamate or succinate but had little effect on 2,4-dinitrophenol-stimulated respiration. 2. 2. The formation of ATP from ADP and phosphate was depressed by DTNB. The inhibition could be reversed by dithiothreitol. 3. 3. The increase in mitochondrial oxygen uptake produced by calcium and phosphate was depressed by DTNB. 4. 4. The uptake of calcium by mitochondria in the presence of ATP and glumate was only partially inhibited by DTNB. In contrast, the stimulatory effect of inorganic phosphate on calcium transport was completely prevented. 5. 5. In the presence of glutamate and no added ATP the uptake of calcium was associated with an entrance of phosphate. DTNB almost abolished the uptake of phosphate and inhibited calcium uptake by about 60%. The decrease in calcium uptake produced by DTNB was equal, mole for mole, to the decrease in phosphate uptake. 6. 6. DTNB completely prevented the extrusion of calcium exhibited by calcium-loaded mitochondria incubated in the presence of inorganic phosphate and a low concentration of magnesium. 7. 7. N-Ethylmaleimide had effects similar to DTNB but in addition severely inhibited 2,4-dinitrophenol-stimulated respiration with glutamate as substrate. 8. 8. It was concluded that a reactive site involving a sulfhydryl group is intimately involved in either the entrance of inorganic phosphate into the mitochondrion or in the formation of a phosphorylated intermediate essential for oxidative phosphorylation and ion transport.