Lill H, Engelbrecht S, Junge W
Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, West Germany.
J Biol Chem. 1988 Oct 5;263(28):14518-22.
The ATP synthase of chloroplasts consists of a proton-conducting portion, CF0, and a catalytic portion, CF1. The smaller subunits of CF1, in particular delta, may play a key role in the coupling of proton transport to ATP synthesis. Purified subunit delta, when added to partially CF1-depleted thylakoid membranes, can restore photophosphorylation (Engelbrecht, S., and Junge, W. (1987) Eur. J. Biochem. 172, 213-218). We report here that it does so by blocking proton conduction through CF0. Thylakoids were CF1-depleted by incubation in hypoosmolar NaCl/EDTA solutions. Variation of the NaCl concentrations and of the incubation times not only changed the overall degree of CF1 depletion but also the subunit composition of solubilized CF1, namely CF1 containing delta and CF1(-delta). This was quantified by immunoelectrophoresis and by fast protein liquid chromatography. Proton conduction was measured by flash spectrophotometry by using standard electrochromic and pH-indicating absorption changes. The removal of integral CF1 was correlated with high electric conductance of thylakoid membranes, an increased extent of rapid proton leakage, and loss of ATP synthesis activity, which exceeded the percentual loss of CF1. The removal of predominantly CF1(-delta) resulted in comparatively lesser effects on the loss of ATP synthesis and on the extent and velocity of proton leakage. On the same line, addition of integral CF1 and of purified delta diminished the electric leak in CF1-depleted thylakoids. Both approaches, the controlled removal of CF1 and CF1(-delta), respectively, and addition of delta and CF1 showed that delta can act as a "stopcock" to the exposed proton channel CF0.
叶绿体的ATP合酶由质子传导部分CF0和催化部分CF1组成。CF1的较小亚基，特别是δ亚基，可能在质子转运与ATP合成的偶联中起关键作用。纯化的δ亚基添加到部分耗尽CF1的类囊体膜中时，可恢复光合磷酸化（恩格尔布雷希特，S.，和荣格，W.（1987年）《欧洲生物化学杂志》172，213 - 218）。我们在此报告，它是通过阻断质子通过CF0的传导来实现的。类囊体通过在低渗NaCl/EDTA溶液中孵育而耗尽CF1。NaCl浓度和孵育时间的变化不仅改变了CF1耗尽的总体程度，还改变了溶解的CF1的亚基组成，即含有δ亚基的CF1和不含δ亚基的CF1（CF1(-δ)）。这通过免疫电泳和快速蛋白质液相色谱进行了定量。通过使用标准的电致变色和pH指示吸收变化的闪光分光光度法测量质子传导。完整CF1的去除与类囊体膜的高电导率、快速质子泄漏程度的增加以及ATP合成活性的丧失相关，后者超过了CF1的百分比损失。主要去除CF1(-δ)对ATP合成损失以及质子泄漏的程度和速度的影响相对较小。同样，添加完整的CF1和纯化的δ亚基可减少耗尽CF1的类囊体中的电泄漏。分别对CF1和CF1(-δ)进行受控去除以及添加δ亚基和CF1这两种方法均表明，δ亚基可作为暴露的质子通道CF0的“旋塞阀”。
