Enrichment and functional reconstitution of glutathione transport activityfrom rabbit kidney mitochondria: Further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport

In previous studies, we provided evidence for uptake of glutathione (GSH) b y the dicarboxylate and the a-oxoglutarate carriers in rat kidney mitochond ria, To investigate further the role of these two carriers, GSH transport a ctivity was enriched from rabbit kidney mitochondria and functionally recon stituted into phospholipid vesicles. Starting with 200 mg of mitoplast prot ein, 2 mg of partially enriched proteins were obtained after Triton X-114 s olubilization and hydroxyapatite chromatography, The reconstituted proteoli posomes catalyzed butylmalonate-sensitive uptake of [C-14]malonate, phenyls uccinate-sensitive uptake of [C-14]2-oxoglutarate, and transport activity w ith [H-3]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), w hich is very close to that previously determined in freshly isolated rat ki dney mitochondria. The enrichment procedure resulted in an approximately 60 -fold increase in the specific activity of GSH transport, Substrates and in hibitors for the dicarboxylate and the a-oxoglutarate carriers (i.e,, malat e, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [H-3]GSH, whereas most substrates for the tricarbox ylate and monocarboxylate carriers had no effect. GSH uptake exhibited an a pparent K-m of 2.8 mM and a V-max of 260 nmol/min per mg protein, Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall G SH uptake and that both carriers account for 70 to 80% of total GSH uptake, These results provide further evidence for the function of the dicarboxyla te and a-oxoglutarate carriers in the mitochondrial transport of GSH. (C) 2 000 Academic Press