Inhibition of Electron and Energy Transfer in Mitochondria

The inhibition by oxybarbiturates of diphosphopyridine nucleotide (DPN)-linked oxidations in mitochondria was observed some time ago (l-4). Their effect on the associated phosphorylation was subsequently reported (5,6), but it is now apparent that oxybarbiturates do not uncouple oxidative phosphorylation (7) nor do they inhibit dinitrophenol-activated adenosine triphosphatase (7). Amytal inhibition of DPNH oxidation has been stated to be insensitive to dinitrophenol (3, 7), and Amytal appears to have no effect on succinoxidase activity (7). Oligomycin (8, 9) and guanidine (10) exhibit some effects similar to those of Amytal, but these are reversible by dinitrophenol (8, 10, 11). This difference in sensitivity to uncoupling agents is found to be a matter of degree and may not represent a significant distinction between sites of action of Amytal and guanidine. In nonphosphorylating preparations in which electron transfer proceeds through the components of the respiratory chain, and not through the Amytaland antimycin-insensitive bypass (12), the site of Amytal inhibition was shown to be on the oxygen side of flavoprotein (12, 13) and has since been localized between flavoprotein and ubiquinone (14).’ A related site involved in succinate oxidation is here found to be sensitive to high concentrations of methylene glycol and of Amytal (11, 15, 16) ; a similar effect of Amytal has been reported for cu-glycerophosphate (17) and choline oxidation (18). In phosphorylating mitochondria, spectroscopic studies show a crossover point for Amytal between flavin and DPNH; .4mytal concentrations required for half-maximal inhibition of both respiratory rate and steady state oxidation levels of the respiratory components have been determined. Somewhat higher concentrations of methylene glycol have similar effects on DPNH oxidation. The object of this paper is to present quantitative data on the inhibition of respiratory activity by Amytal inoxidation not only of DPN-linked substrates but of succinate as well. That the effectiveness of Amytal is less in the presence of uncoupling agents than in the presence of adenosine diphosphate (ADP) and phosphate indicates an inhibition of energy transfer in addition to an inhibition of electron transfer, The spectroscopic effects of Amytal are studied with particular emphasis upon the responses of cytochrome b and pyridine nucleotide, which are especially useful in comparisons between the effects of Amytal