Subunit structure and ion transport mechanism of vacuolar ATPase

(1)成功大规模纯化了产肠球菌v - atp酶，实现了柱层析；从20升培养基中获得约30毫克的纯化酶，包括9个亚基，A， B， C， D， E， F， G， I， K。SDS-PAGE结果显示，A、B、C、D、E、F和G 7个亚基与EDTA孵育后可作为V1亚基释放，形成V0V1复合物；V0部分由I和K个亚基组成。（ii） E.hirae v -ATP酶催化Na+或Li+易位并偶联ATP水解。这表明该多亚基酶的NtpK蛋白脂亚基的谷氨酸残基（Glu139）是这些离子进行易位的结合位点。我们建立了ntpK基因与其缺失突变体的互补系统，发现将Glu139替换为天冬氨酸后，ATPase活性消失。因此，NtpK酸性残基的侧链长度对atp酶反应非常重要。（iii）我们研究了E.hirae Na+易位atp酶的离子结合步骤。Na+与纯化V-ATPase的结合动力学表明6个Na+结合/酶分子具有单一高亲和力（Kd (Na+) =15微米）。阳离子结合位点的数量与V-ATPase蛋白脂形成由六聚体组成的转子环的模型一致，每个六聚体具有一个阳离子结合位点。这表明Na+- atp酶的Na+结合位点很容易从水相中获得。

(i) We succeeded in the large-scale purification of Enterococcus hirae V-ATPase achieving column chromatographies ; about 30 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, K, was obtained from 20 liter culture. SDS-PAGE revealed that seven subunits, A, B, C, D, E, F and G, are releasable as the V1 subunit form thew V0V1 complex by incubation with EDTA ; the V0 portion consists of I and K subunits.(ii) E.hirae V-ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. We established a complementation system from the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 to aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.(iii) We investigated the ion binding step to E.hirae Na+-translocating ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 Na+ bound/enzyme molecule, with a single high affinity (Kd (Na+) =15 microM.The number of cation binding sites is consisitent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. This suggest that the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase.