Mg^（2＋）促进线粒体H~＋－ATP酶的重建机理──H~＋－ATP酶复合体亚基水平的研究

MECHANISM OF Mg￣(2+) EFFECT ON RECONSTITUTION OF MITOCHONDRIAL H￣+-ATPase ──STUDIED WITH SUBUNITS OF THE ATPase COMPLEX

用专一性标记蛋白质巯基（－ＳＨ）的荧光探剂ａｃｒｙｌｏｄａｎ测定含Ｍｇ２＋的Ｆ０－ＡＴＰ酶或Ｆ０－ＯＳＣＰ－Ｆ１－ＡＴＰ酶的脂酶体的构象与无Ｍｇ２＋者明显不同，前者的蛋白质的－ＳＨ基团处于疏水性更强的微环境中；在有Ｍｇ２＋和ＯＳＣＰ同时存在下重建的Ｆ０－Ｆ１－ＡＴＰ酶脂酶体较无ＯＳＣＰ者表现更高的水解活力或膜电位，表明ＯＳＣＰ增强Ｍｇ２＋的促进作用，这进一步提示Ｍｇ２＋通过改变膜脂的物理状态促进线粒体Ｈ＋－ＡＴＰ酶重建的间接作用。这些实验结果，从线粒体Ｈ＋－ＡＴＰ酶复合体的亚基水平的相关性上，对于我们提出的Ｍｇ２＋通过改变膜脂的物理状态使之具有合适的流动性，诱导嵌入脂双层的Ｈ＋－ＡＴＰ酶复合体的Ｆ０的构象发生变化并传递至复合体的催化中心Ｆ１，从而使重建Ｆ１－Ｆ０－ＡＴＰ酶具有较适合的蛋白构象，表现较高的重建酶活性的假设提供了直接的实验证据，精确地阐明了Ｍｇ２＋促进线粒体Ｆ０－Ｆ１－ＡＴＰ酶重建作用的分子机理。

A conformational difference of the F0- ATPase or F0-F1- ATPase- containing proteoliposomes by induced Mg2+ effect could be detered using acrylodan, a sensitive probe for detecting-SH groups in proteins. -SH groups in the L F0-ATPase with Mg2+ or L(F0 + F1)-ATPase proteoliposomes with Mg2+ and OSCP were deeply buried in more hydrophobic environment compared with that without Mg2+ or with Mg2+ but no OSCP, respectively .ATPase activity or membrane potential (△ψ) of the F0-F1-ATPase reconstituted in the presence of both Mg2+ and OSCP were significantly higher than that only in the presence of Mg2+, which may indicate that OSCP further stimulated the reconstitution of Mg2+ -containing F0-F1-ATPase. The results described above provide a direct evidence for our hypothesis that Mg2+-mediated alteration in lipid fluidity induces a conformational change in the F0 portion which is in turn transmitted by OSCP to the F1 sector, resulting in formation of a suitable conformation of the F0-F1-ATPase with higher activities.