Kinetic study of the dicarboxylate carrier in rat liver mitochondria.

Abstract

The rate of uptake in mitochondria as catalyzed by the dicarboxylate carrier of dicarboxylates and, in the presence of N‐ethylmaleimide, of inorganic phosphate, has been analyzed by using the inhibitor stop method. By comparing the dependence on substrate concentration of the rate of succinate, malonate and malate uptake, it is found that the Km (malate) = 0.23 < Km (malonate) = 0.37 < Km (succinate) = 1.17 mM. The V values, however, are not significantly different for all three dicarboxylates (V∼ 70 μmoles/g protein × min at 9°C). Succinate, malate and malonate are shown to be competitive with each other in the kinetics of uptake. The inhibition of the rate of malate uptake by 2–phenylsuccinate is found to be competitive. Several “permeant” anionic substrates such as acetate, pyruvate, 3‐hydroxybutyrate, glutamate and aspartate have no effect on the rate of dicarboxylate uptake, even when added in large excess. The rate of malonate uptake is inhibited by 2‐oxoglutarate but unaffected by citrate, while the rates of succinate and malate uptake are decreased by both 2‐oxoglutarate and citrate. Malate is inhibited more than succinate. The inhibition of the rate of malate uptake by citrate and 2‐oxoglutarate is shown to be non‐competitive. This is explained due to a re‐exchange of accumulated malate by citrate or 2‐oxoglutarate. In the presence of N‐ethylmaleimide, externally added Pi inhibits the rate of dicarboxylate uptake. The Pi inhibition of malate uptake is found to be mixed with a larger share of non‐competitive than competitive inhibition. The V of the rate of P1 uptake is approximately equal to that of malate uptake in the presence of N‐ethylmaleimide. The K1 is, however, higher for Pi (Ki=Kp= 1.95 mM) than for malate. It is concluded firstly that the dicarboxylate carrier specifically transports either a dicarboxylate or Pi (but not both together), and secondly that the carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates.