大鼠心脏线粒体支链α-酮酸转运体的增溶与功能重建
Solubilization and functional reconstitution of the branched-chain alpha-keto acid transporter from rat heart mitochondria.
作者信息
Hutson S M, Roten S, Kaplan R S
机构信息
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103.
出版信息
Proc Natl Acad Sci U S A. 1990 Feb;87(3):1028-31. doi: 10.1073/pnas.87.3.1028.
The mitochondrial branched-chain alpha-keto acid transporter was solubilized from rat heart mitochondria and its function was reconstituted in phospholipid vesicles. The transporter was extracted from mitoplasts with Triton X-114 in the presence of exogenous cardiolipin and alpha-ketoisocaproate. Upon incorporation of this extract into asolectin vesicles by the freeze-thaw-sonication technique, a p-chloromercuribenzoate-sensitive, protein-dependent transport of alpha-ketoisocaproate into the proteoliposomes was observed. Significant inhibition of alpha-ketoisocaproate transport was observed in the reconstituted system with branched-chain alpha-keto acids (64-83%) and the related carboxylates alpha-ketocaproate (58%) and alpha-ketovalerate (49%), but not with substrates for the pyruvate carrier (less than 5%). The reconstituted carrier was substantially inhibited by sulfhydryl reagents, by the histidine-specific reagent diethyl pyrocarbonate, and by the tyrosine-specific reagent N-acetylimidazole. The extraction and functional reconstitution of the branched-chain alpha-keto acid transporter represents an important first step towards purification and molecular characterization of this anion carrier.
线粒体支链α-酮酸转运体从大鼠心脏线粒体中溶解出来，并在磷脂囊泡中重建其功能。在存在外源性心磷脂和α-酮异己酸的情况下，用Triton X-114从线粒体膜间颗粒中提取该转运体。通过冻融超声处理技术将该提取物掺入大豆卵磷脂囊泡后，观察到对氯汞苯甲酸敏感的、蛋白质依赖性的α-酮异己酸转运到蛋白脂质体中。在重建系统中，支链α-酮酸（64-83%）、相关羧酸盐α-酮己酸（58%）和α-酮戊酸（49%）对α-酮异己酸转运有显著抑制作用，但丙酮酸载体的底物则无此作用（小于5%）。重建的载体受到巯基试剂、组氨酸特异性试剂焦碳酸二乙酯和酪氨酸特异性试剂N-乙酰咪唑的显著抑制。支链α-酮酸转运体的提取和功能重建是该阴离子载体纯化和分子表征的重要第一步。
Zotero 插件