Mg^(2+)对线粒体H^+-ATP酶的F_O在脂质体重建时的影响

EFFECT OF Mg^(2-) ON THE H^+- TRANSLOCATION ACTIVITY OF RECONSTITUTED MITOCHONDRIAL F_0-ATPase

线粒体ATP合成酶是由具有H^+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg^(2+)和无Mg^(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg^(2+)条件下形成的脂酶体(L·F_0)+Mg^(2+)较无Mg^(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH^+′sF_0,后者为3nmolH^+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg^(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H^+—AhP酶具有较高活性的假设.

Mitochondrial ATP synthase consists of a membrane - integrated part, F0 that constitutes a trans-membrane proton channel, and a water - soluble extrinsic part. F1 that carries the catalytic center for interconversion of ATP and ADP. Purification of F0 segment from H+-ATPase complex of porcine heart mitochondria was carried out by enzyme - extracting with NaBr and simple sonication. Reconstitution of the purified F0 on asolectin liposomes was performed by cholate dialysis method in the absence or presence of Mg2+(lmmol/L). H+ -translocation of the proteoliposomes (L · F0) with or without Mg2+ was monitored by measuring fluorescence quenching of 9-aminoacridine (9 -AA) probe and pH change using pH meter equipped with complex electrode or a fast -response pH -ISFET electrode. The data showed that fluorescence quenching of 9-AA for L · F11 with Mg2+ was ca. 30% higher than that of L · F11 without Mg2+. The initial rate of H+-translocation of the former was 5 nmol H+s · nmol F0, and 3 for the latter, i.e. about one fold higher of the H+-translocation activity for L · F0 with Mg2+. The results obtained by the two approaches identically indicated that H+-translocation activity for Mg2+-containing L · F0 was significantly higher than that of Mg2+ -free L · F0. This may imply that Mg2+-mediated change in physical state of lipids in the proteoliposomes would favor formation of a suitable conformation of the reconstitued F0 with higher H+-translocation activity. Obtained results may provide further evidence for our previous hypothetic scheme that Mg2+-mediated altering in lipid fluidity induces a comformational and activity change of F0 and F1.