Kinetic Study of the Tricarboxylate Carrier in Rat Liver Mitochondria

The kinetics of citrate uptake by malate‐loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms.1 The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min −1 and the corresponding rate of citrate uptake of 13.4 μmol × min −1 × g protein −1 (at 0.5 mM citrate and 9 °C). The half time is 34 sec. 2 The temperature dependence of citrate exchange was measured in the range between 0 and 14 °C. An activation energy of 20.1 kcal and a Q 10 of 3.6 can be calculated from the Arrhenius plot. 3 The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The K m and V values for the rate of citrate uptake are 0.12 ± 0.01 mM and 22.5 ± 1.8 μmol citrate × min −1 × g protein −1 respectively at 9 °C in 11 experiments. 4 The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH. 5 The rate of citrate exchange is inhibited in a competitive manner by cis ‐aconitate, threo ‐D s ‐isocitrate, 1,2,3‐propanetricarboxylate, 1,2,3‐benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans ‐aconitate and 1,3,5‐pentanetricarboxylate have no effect on citrate exchange, even when added in large excess. 6 Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2‐oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is greater with malate than with succinate. 7 The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specific inhibitors of the dicarboxylate carrier. 8 The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates ( K 1 is 0.7 mM for malate). 9 Phospho enol pyruvate strongly inhibits the rate of citrate uptake, while P 1 and pyruvate have only a slight effect. The inhibition by phospho enol pyruvate is shown to be competitive ( K 1 is 0.11 mM). 10 It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, phospho enol pyruvate and dicarboxylates. The implications of these findings are discussed.