Kinetics and Specificity of the Oxoglutarate Carrier in Rat‐Liver Mitochondria

Abstract

The kinetics of the oxoglutarate/malate exchange have been measured and the specificity of the oxoglutarate carrier has been investigated in rat liver mitochondria. Oxoglutarate is taken up by malate‐loaded mitochondria at a rate of 33.2 μmol/min per g protein (at 0.25 mM oxoglutarate and 9°C). The uptake follows a first order kinetics with a kof 2.13 min−1 and t1/2 of 32 s. An EA of 20.5 kcal and a Q10 of 3.6 are obtained from the study of the temperature‐dependence of oxoglutarate exchange. The Km and V values for the rate of oxoglutarate uptake have been evaluated from Line‐weaver‐Burk plots under different conditions. At 9°C and at pH 7.0, Km, is 46.0 ± 1.2 μM and F is 42.8 ± 2.2 μmol/min per g protein (mean of 10 experiments). The rate of oxoglutarate uptake increases, as the pH decreases. The inhibition by higher pH is competitive with oxoglutarate. In a low ionic strength sucrose medium, cations stimulate the rate of oxoglutarate uptake. This activation is of the competitive type. The rate of oxoglutarate uptake is inhibited by several non substituted dicarboxylates, 2‐oxo‐, 2‐hydroxy‐ and 2‐aminodicarboxylates. The nonpenetrant dicarboxylate analogues, such as butylmalonate and phenylsuccinate, previously thought to be specific inhibitors of the dicarboxylate carrier, inhibit also oxoglutarate uptake. The inhibition of the rate of oxoglutarate uptake by dicarboxylates and their analogues is found to be competitive. Among the dicarboxylates tested, malate has the highest affinity (K1 of 0.12 mM). The oxodicarboxylate oxaloacetate has a K1 higher than that of malate (K1 is 1.1 mM). Several monocarboxylates, oxo‐ and amino acids have no effect on the rate of oxoglutarate uptake, even when added in large excess. The rate of oxoglutarate exchange is also inhibited in a competitive manner by several tricarboxylates. The affinity for tricarboxylates is, however, very low [Km (citrate) is 3.6 mM]. In oxoglutarate‐loaded mitochondria, several dicarboxylates, but not tricarboxylates, are able to exchange with oxoglutarate. The exchange between aminodicarboxylates and oxoglutarate is insensitive to phenylsuccinate. Furthermore glutamate and aspartate do not exchange with intramitochondrial malate or malonate. It is concluded that the oxoglutarate carrier has a single binding site for oxoglutarate and for several other dicarboxylates, some of which are translocated by this carrier. Tricarboxylates, at high concentrations, block the carrier by attaching to the substrate‐binding site without being transported. The implications of these findings are discussed. Copyright © 1972, Wiley Blackwell. All rights reserved