Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: Further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport
Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: Further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport
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DOI：
10.1006/abbi.1999.1527
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发表时间：
2000-01-01
影响因子：
3.9
通讯作者：
Lash, LH
中科院分区：
文献类型：
--
作者：
Chen, ZF;Putt, DA;Lash, LH
在之前的研究中，我们提供了证据，证明谷胱甘肽（GSH）被二羧酸盐和a-氧葡萄糖酸盐载体在大鼠肾脏线粒体中摄取，为了进一步研究这两种载体的作用，我们从兔肾脏线粒体中增强了谷胱甘肽的运输活性，并在功能上重构为磷脂囊泡。从200 mg有丝分裂体蛋白开始，经Triton X-114增溶和羟基磷灰石层析得到2 mg部分富集的蛋白，重组的蛋白脂质体催化丙二酸丁酯对[C-14]丙二酸的敏感摄取，苯基琥珀酸对[C-14]2-氧戊二酸的敏感摄取，以及与[H-3]GSH的转运活性。5 mM谷胱甘肽的初始摄取速率约为170 nmol/min / mg蛋白质，一级速率常数为0.3 min(-1)，这与先前在新分离的大鼠肾脏线粒体中测定的非常接近。富集过程导致GSH运输的比活性增加了大约60倍，二羧酸盐和a-氧戊二酸盐载体（即苹果酸盐、丙二酸盐、2-氧戊二酸盐、丁基丙二酸盐、苯琥珀酸盐）的底物和抑制剂显著抑制了[H-3]GSH的摄取，而三羧酸盐和单羧酸盐载体的大多数底物没有影响。GSH的表观K-m为2.8 mM， V-max为260 nmol/min / mg蛋白，GSH与二羧酸盐的相互抑制分析表明，二羧酸盐载体对GSH的总摄取比例较高，占GSH总摄取的70 ~ 80%，这些结果进一步证明了二羧酸盐和a-氧葡萄糖酸盐载体在GSH的线粒体运输中的作用。(C) 2000年学术出版社
In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the a-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 [C-14]malonate, phenylsuccinate-sensitive uptake of [C-14]2-oxoglutarate, and transport activity with [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), 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 a-oxoglutarate carriers (i.e,, malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [H-3]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 a-oxoglutarate carriers in the mitochondrial transport of GSH. (C) 2000 Academic Press