Chen Z, Putt D A, Lash L H
Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan, 48201, USA.
Arch Biochem Biophys. 2000 Jan 1;373(1):193-202. doi: 10.1006/abbi.1999.1527.
In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]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), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent 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 GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.
在先前的研究中，我们提供了大鼠肾线粒体中二羧酸和2-氧代戊二酸载体摄取谷胱甘肽（GSH）的证据。为了进一步研究这两种载体的作用，从兔肾线粒体中富集了GSH转运活性，并将其功能性重组到磷脂囊泡中。以200mg线粒体质蛋白为起始材料，经Triton X-114溶解和羟基磷灰石层析后，获得了2mg部分富集的蛋白。重组的蛋白脂质体催化了对[（14）C]丙二酸的丁基丙二酸敏感摄取、对[（14）C]2-氧代戊二酸的苯基琥珀酸敏感摄取以及与[（3）H]GSH的转运活性。5mM GSH的初始摄取速率约为每毫克蛋白170nmol/min，一级速率常数为0.3min（-1），这与先前在新鲜分离的大鼠肾线粒体中测定的结果非常接近。富集过程使GSH转运的比活性增加了约60倍。二羧酸和2-氧代戊二酸载体的底物和抑制剂（即苹果酸、丙二酸、2-氧代戊二酸、丁基丙二酸、苯基琥珀酸）显著抑制了[（3）H]GSH的摄取，而三羧酸和单羧酸载体的大多数底物则没有影响。GSH摄取的表观K（m）为2.8mM，V（max）为每毫克蛋白260nmol/min。GSH与二羧酸之间的相互抑制分析表明，二羧酸载体对总体GSH摄取的贡献比例略高，并且两种载体占总GSH摄取的70%至80%。这些结果为二羧酸和2-氧代戊二酸载体在线粒体GSH转运中的功能提供了进一步的证据。
