Hirata Tomoyuki, Iwamoto-Kihara Atsuko, Sun-Wada Ge-Hong, Okajima Toshihide, Wada Yoh, Futai Masamitsu
Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.
J Biol Chem. 2003 Jun 27;278(26):23714-9. doi: 10.1074/jbc.M302756200. Epub 2003 Apr 1.
Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.
液泡型ATP酶V1V0（V-ATP酶）普遍存在于真核细胞的内膜细胞器中。在本研究中，我们分别在酿酒酵母V-ATP酶的c亚基和G亚基上通过基因工程引入了His标签和生物素标签。利用这种工程酶，当该酶通过c亚基固定在玻璃表面时，我们直接观察到附着在G亚基上的肌动蛋白丝持续逆时针旋转。V-ATP酶产生的扭矩与F-ATP酶（ATP合酶）基本相同。这种旋转受到抗霉素和硝酸盐的抑制，但不受叠氮化物的抑制。这些结果表明，V-ATP酶和F-ATP酶进行共同的旋转催化。
