Mechanism of the Exchanges Catalysed by the Oxoglutarate Translocator of Rat‐Heart Mitochondria

Abstract

The kinetics of the exchange reactions between 2‐oxoglutarate, malate and malonate have been measured at 4 °C in preparations of rat‐heart mitochondria under conditions where the oxoglutarate translocator is operating exclusively. This was possible since no activity of the tricarboxylate translocator can be measured in such preparations and since the activities of the phosphate and dicarboxylate translocators may be blocked by mersalyl, to which the oxoglutarate translocator is insensitive. The metabolism of the substrates was prevented by addition of rotenone and arsenite. Measurements were made at three different external and three different internal concentrations of the substrates. The results show that the affinity of the oxoglutarate translocator is very much higher for external substrates than for the corresponding internal substrates. The Michaelis constants increase in the following order: external oxoglutarate < external malate < external malonate < internal oxoglutarate < internal malate < internal malonate. The results also show that the affinity of the translocator for a given substrate, either external or internal, is independent of the nature of the counter‐ion. This is not true of the maximal velocities of the exchange reactions. Of the various mechanism described by Cleland for two‐substrate‐two‐product enzyme‐catalysed reactions, only the so‐called mechanism of rapid‐equilibrium random bi‐bi is compatible with our experimental results. This implies that the Michaelis constants determined are the dissociation constants of the various translocator‐substrate(s) complexes.