Transport of Dicarboxylic Acids in Castor Bean Mitochondria

Publisher Website
Google Scholar
Abstract
Mitochondria from castor bean (R. communis cv. Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of 14C from [14C]malate or [14C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of 14C from [14C]malate or [14C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent Km values of 2-2.5 mM. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin, but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 mM. CTP was effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate. The maximum rate of succinate accumulation (14.5 nmol .cntdot. min-1mg protein) was only a fraction of the measured rate of oxidation (100-200 nmol .cntdot. min-1/mg protein). Efflux of malate from the mitochondria occurred at high rates (150 nmol per .cntdot. min-1/mg protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [14C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence f phosphate, uptake of [14C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [14C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [14C]succinate into mitochondria with malate was rapid 27 nmol/15 s .cntdot. mg-1 protein at 4.degree. C) and inhibited by butylmalonate, benzylmalonate and phenylsuccinate. Uptake of [14C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent Km of 2.5 mmol and Vmax of 250 nmol .cntdot. min-1/mg protein at 4.degree. C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.