Sanadi D R, Pringle M, Kantham L, Hughes J B, Srivastava A
Proc Natl Acad Sci U S A. 1984 Mar;81(5):1371-4. doi: 10.1073/pnas.81.5.1371.
Membrane energization by ATP has been measured in vesicles containing purified bovine heart mitochondrial H+-ATPase (ATP synthase) with the voltage-sensitive dye oxonol VI. The dithiol chelator, Cd2+, and the thiol oxidant, copper o-phenanthroline, produced discharge of the membrane potential when added at the steady state and inhibited its establishment when added prior to energization by ATP. These effects, which were reversed by dithiothreitol, were not accompanied by an increase in the nonspecific H+ permeability of the membrane. Passive H+ conduction in proteoliposomes containing F0 (hydrophobic segment of ATP synthase) was assayed by the quenching of 9-aminoacridine fluorescence after establishing a K+ diffusion potential. This conductance was blocked by Cd2+, an inhibitor of coupling factor B (FB). Labeling of F0 with 115Cd2+ at the concentrations that inhibited the F0 conductance followed by gel electrophoresis yielded a single radioactive band with a molecular weight corresponding to FB, the presence of which in the F0 preparation was confirmed by immunoblot staining. The data offer strong evidence that FB is an essential component of the H+ channel of F0, because H+ conduction through the channel is inhibited by chemical modification of FB.
利用电压敏感染料氧化芪三酚 VI，在含有纯化牛心线粒体 H⁺ -ATP 酶（ATP 合酶）的囊泡中测定了 ATP 对膜的能量化作用。二硫醇螯合剂 Cd²⁺和硫醇氧化剂邻菲罗啉铜，在稳态时添加会导致膜电位放电，在 ATP 能量化之前添加则会抑制膜电位的建立。这些作用可被二硫苏糖醇逆转，且并未伴随着膜非特异性 H⁺通透性的增加。在建立 K⁺扩散电位后，通过 9 -氨基吖啶荧光猝灭来测定含有 F0（ATP 合酶的疏水片段）的蛋白脂质体中的被动 H⁺传导。这种传导被耦合因子 B（FB）的抑制剂 Cd²⁺所阻断。在抑制 F0 传导的浓度下用¹¹⁵Cd²⁺标记 F0，随后进行凝胶电泳，得到一条单一的放射性条带，其分子量与 FB 相对应，通过免疫印迹染色证实了 F0 制剂中存在 FB。这些数据提供了有力证据，表明 FB 是 F0 的 H⁺通道的重要组成部分，因为通过该通道的 H⁺传导会因 FB 的化学修饰而受到抑制。
