Studies of the energy-transfer system of submitochondrial particles. Kinetic studies of the effect of oligomycin on the respiratory chain of EDTA particles.

Abstract

1 Oligomycin induces an inhibition of respiration in EDTA particles from beef-heart mitochondria which is relieved by uncouplers. The “respiratory control”, measured as the respiratory stimulation caused by the addition of uncoupler to the oligomycin-supplemented system, is as an average 3-fold in the case of NADH, and 2-fold in the case of succinate as substrate. Little or no inhibition of respiration by oligomycin is observed with ascorbate +N,N,N′,N′ -tetramethyl-p -phenylenediamine or phenazine methosulfate as substrate. 2 Partial inhibition of NADH oxidase with rotenone, antimycin A, azide, or cyanide does not alter the extent of stimulation of the oligomycin-inhibited respiration by uncoupler. However, when NADH oxidase is limited by an externally added NADH-generating system, such as ethanol + alcohol dehydrogenase or glutamate + glutamate dehydrogenase, respiratory control is diminished; this effect is less pronounced when NADH is generated by means of NADPH and the pyridine nucleotide transhydrogenase present in the particles. Likewise, the extent of stimulation of the oligomycin-inhibited respiration by uncoupler remains unchanged when succinoxidase is limited by antimycin A, but is decreased when it is limited by malonate. 3 The apparent rate constants derived from steady state analysis of cytochromes for the sequential reactions of the NADH and succinate oxidase systems are increased 2–4-fold by adding uncoupler to the oligomycin-inhibited system for all steps except succinate-cytochrome b. 4 In the presence of partially inhibitory concentrations of cyanide, all cytochromes show biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. With succinate as substrate, the reduction is biphasic in the case of cytochromes c and a, but not in the case of cytochrome b. Uncoupler abolishes the biphasic character of cytochrome reduction, which now shows rapid first-order kinetics. 5 Similarities and differences between the features of the control of electron transport by energy-conserving mechanisms as found with intact mitochondria and submitochondrial particles are discussed. The usefulness of EDTA particles for the study of some qualitative and quantitative aspects of the energized state of mitochondria is pointed out.