Effect of Phosphate and Uncouplers on Substrate Transport and Oxidation by Isolated Corn Mitochondria

Abstract

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O(2) uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K(+) permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V(max), probably reflecting a diffusion-limited process.The results indicate that rapid malate and succinate oxidation require the operation of both the phosphate and dicarboxylate transporters, which in turn depend on maintenance of a proton motive force across the inner membrane. In addition, phosphate can stimulate acceptorless malate oxidation by reaction with the coupling mechanism, and in uncoupled mitochondria which are depleted of substrate there is a slow rate of oxidation which appears to be limited by diffusive entry.