EFFECT OF Mg~(2-) ON THE H~+- TRANSLOCATION ACTIVITY OF RECONSTITUTED MITOCHONDRIAL F_0-ATPase
Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

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.