Connern C P, Halestrap A P
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochem J. 1994 Sep 1;302 ( Pt 2)(Pt 2):321-4. doi: 10.1042/bj3020321.
Binding of mitochondrial matrix cyclophilin (CyP) to the rat liver mitochondrial membranes was detected by SDS/PAGE and Western blotting with suitable antipeptide antibodies. Binding was not affected by prior exposure of mitochondria to Ca2+, adenine nucleotides or inhibitors of the adenine nucleotide translocase, but was greatly increased by t-butyl hydroperoxide (tBH), phenylarsine oxide or diamide. These all sensitized the opening of the non-specific mitochondrial pore to [Ca2+], and the effect of tBH was shown to be maintained after washing away the tBH, consistent with it being caused by the enhanced CyP binding. The bound CyP did not demonstrate peptidyl-prolyl cis-trans isomerase activity. CyP-binding was prevented by 5 microM cyclosporin A, but not reversed by cyclosporin treatment of the membranes. The effect of tBH on binding was concentration-dependent and maximal within 30 s.
通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）以及使用合适的抗肽抗体进行蛋白质免疫印迹法，检测到线粒体基质亲环蛋白（CyP）与大鼠肝脏线粒体膜的结合。线粒体预先暴露于钙离子、腺嘌呤核苷酸或腺嘌呤核苷酸转位酶抑制剂中，结合不受影响，但叔丁基过氧化氢（tBH）、苯砷酸氧化物或二酰胺可使其结合显著增加。这些物质均使非特异性线粒体孔对钙离子的开放敏感性增强，并且在洗去叔丁基过氧化氢后，其作用仍能维持，这与它是由增强的亲环蛋白结合所引起的一致。结合的亲环蛋白未表现出肽基脯氨酰顺反异构酶活性。5微摩尔环孢素A可阻止亲环蛋白结合，但对经环孢素处理的膜不能使其结合逆转。叔丁基过氧化氢对结合的影响呈浓度依赖性，且在30秒内达到最大值。
