Abstract
Effects of inhibitors of phosphorylation on the oxidation of succinate and of l-malate were investigated with tightly coupled mitochondria isolated from mung bean hypocotyls. When mitochondria were incubated with 2,4-dinitrophenol, or carbonyl cyanide m-chlorophenylhydrazone prior to the addition of substrate, the uncoupling effects of these chemicals were relatively small. This is probably caused by relative lack in these mitochondria of endogenous substrates, ATP, and/or "high-energy intermediates". The action of uncoupling agents is, therefore, revealed in a more striking manner when they are introduced during the second state 4. Of the 2 uncoupling agents tested, malate oxidation consistently required 1.5 to 2 times higher concentration of the agents for the half-maximal effects than succinate oxidation. From the comparison of the degree of uncoupling it is concluded that 2,4-dinitrophenol is a better uncoupler of succinate oxidation, whereas carbonyl cyanide m-chlorophenylhydrazone functions as a more complete uncoupler of malate oxidation.Oligomycin does not inhibit state 4 rates, while the increment of respiration due to added ADP is completely inhibited by this antibiotic. Identical half-maximal effects are observed with the same concentration of oligomycin in both succinate and l-malate oxidation. The oligomycin effect depends on the mitochondrial concentration employed. The concentration of this chemical required for the half-maximal effect is 55 to 80 mmumoles per mg mitochondrial protein. It is suggested that this inhibitor of phosphorylation binds all of the phosphorylation sites regardless of whether the sites are functional or not.