Negrin R S, Foster D L, Fillingame R H
J Biol Chem. 1980 Jun 25;255(12):5643-8.
The intrinsic membrane sector (Fo) of the H+-ATPase complex of Escherichia coli has been purified, incorporated into liposomes, and its proton-translocating activity reconstituted. The Fo sector was prepared by treating a purified, particulate, F1FO-ATPase preparation with EDTA to solubilize the F1-ATPase. The resulting particulate Fo fraction was incorporated into liposomes of E. coli phospholipids by sonication. Proton efflux from these liposomes was measured with a pH electrode after imposition of a membrane potential. The kinetics of proton efflux fits that predicted by the Goldman-flux equation. The rate of proton efflux was increased maximally more than 100-fold on incorporation of the Fo sector into the liposomes. The rate of H+ efflux varied directly with the amount of Fo material added during reconstitution. Dicyclohexylcarbodiimide blocked Fo-mediated H+ efflux. Inhibition was shown to be due to reaction of dicyclohexylcarbodiimide with a specific proteolipid subunit of Fo. The preparation of Fo used in these studies contained the three proteins that had previously been identified as likely subunits of Fo (Foster, D. L., and Fillingame, R. H. (1979) J. Biol. Chem. 254, 8230-8236). It remains to be determined whether all three components are required for reconstitution of proton translocation activity.
大肠杆菌H⁺ -ATP酶复合体的内在膜区（F₀）已被纯化，整合到脂质体中，并重建了其质子转运活性。通过用乙二胺四乙酸（EDTA）处理纯化的颗粒状F₁F₀ -ATP酶制剂以溶解F₁ -ATP酶来制备F₀区。通过超声处理将所得的颗粒状F₀部分整合到大肠杆菌磷脂脂质体中。施加膜电位后，用pH电极测量这些脂质体中的质子外流。质子外流动力学符合戈德曼通量方程预测的结果。将F₀区整合到脂质体中后，质子外流速率最大增加了100多倍。H⁺外流速率与重建过程中添加的F₀物质的量直接相关。二环己基碳二亚胺阻断了F₀介导的H⁺外流。已证明抑制作用是由于二环己基碳二亚胺与F₀的一种特定蛋白脂质亚基发生反应。这些研究中使用的F₀制剂包含先前被确定可能是F₀亚基的三种蛋白质（福斯特，D.L.，和菲林盖姆，R.H.（1979年）《生物化学杂志》254，8230 - 8236）。质子转运活性重建是否需要所有这三种成分仍有待确定。
