Activation and inhibition of succinate oxidation following adenosine diphosphate supplements to pigeon heart mitochondria.

The initiation of electron transfer by addition of substrate to nonphosphorylating preparations has been recorded by a number of workers and is generally accepted to be a rapid reaction. Polarographic measurements of oxygen utilization on addition of succinate to Keilin and Hartree heart muscle preparations and spectrophotometric recordings of fumarate formation in the same system support this conclusion (1). However, evidence for rapid initiation of electron transfer on addition of reduced diphosphopyridine nucleotide to Keilin and Hartree preparations is unclear; Slat&s data for such experiments suggest an induction period in the initiation of respiration (2). Respiratory inhibition can occur in nonphosphorylating preparations after oxidation of an appreciable amount of succinate; for example, oxaloacetate may be formed during succinate oxidation provided that fumarase and malic dehydrogenase activities are present and that the latter is supplemented with diphosphopyridine nucleotide (3-5). In phosphorylating mitochondria, the conditions for forming oxaloacetate also exist, since fumarase, malic dehydrogenase, and DPN are present, and it has been known for nearly a quarter of a century that such oxaloacetate inhibition can readily be observed. Actually, it is rather remarkable that succinate canbe oxidized for a continuous interval in various types of mitochondria without inhibition from the formation of oxaloacetate. Inhibition is readily observed, however, if the production of adenosine triphosphate is simultaneously inhibited. The reactivation of electron transfer in such an inhibited system by addition of ATP and the subsequent formation of phosphopyruvate have been reported (6); appropriate conditions for forming high concentrations of phosphopyruvate with mitochondria treated with dinitrophenol involve a requirement for dinitrophenol-insensitive phosphorylation in the oxidation of cu-ketoglutarate (7). The whole question of metabolic regulation by the oxaloacetate level has been considered by Tyler (8). There have also been a number of recent reports of studies in which preincubation of the mitochondria with uncoupling agents caused an inhibition of succinate oxidation (9-12). Such experiments led some investigators to postulate an energy requirement for succinate oxidation (ll-13), and others to attribute the effects to oxaloacetate inhibition (9,10). The latter explanation is being studied by determinations of oxaloacetate (14, 15). Proceeding along different lines, we have observed a delay in