KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT BRAIN MITOCHONDRIA

— The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non‐synaptosomal mitochondria, either on a glutamate‐hydroxyl antiporter or on a glutamate‐aspartate antiporter. An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N ‐ethylmaleimide. Swelling of the mitochondria in iso‐osmotic NH4 + ‐ l ‐glutamate was inhibited in the presence of avenaciolide and N ‐ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino‐oxyacetic acid had no effect on the glutamate‐hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double‐beam spectrophotometry, and this reduction was inhibited on the one hand by N ‐ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of l ‐[ 14 C]glutamate into brain mitochondria was performed by using the centrifugation‐stop procedure. This penetration followed saturation kinetics, with a mean apparent K m of 1.56 M M at pH 7.4 and at 20°C, the value of Knax was 4.34 nmol per min per mg protein in the same conditions. IV‐Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non‐competitive with a K i of 0.7 M m ‐at pH 7.4 and at 20°C. The entry of glutamate was pH‐dependent and it increased 2‐fold in the pH range of 7.4 to 6.4. A temperature‐dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated E A of 12.8 kCal per mol of glutamate and a Q 10 of 2.16. The activity of γ‐glutamyl transpeptidase was practically absent in these rat brain mitochondria. Oxidation of extramitochondrial NADH by the‘malate‐aspartate shuttle’reconstituted in vitro was followed in rat brain non‐synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the ‘shuttle’ did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or γ‐aminobutyrate did not replace glutamate efficiently. A high inhibition of the‘malate‐aspartate shuttle’occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of n ‐ethylmaleimide, glisoxepide or n ‐butylmalonate. Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.