Zakharov S D, Li X, Red'ko T P, Dilley R A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
J Bioenerg Biomembr. 1996 Dec;28(6):483-94. doi: 10.1007/BF02110438.
The 8-kDa subunit c of the E. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mutants, Asp61-->Asn and Asp61-->Gly. The Ca++ binding is pH dependent in both the E. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation. The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.
在将蛋白质从SDS - PAGE凝胶转移到聚偏二氟乙烯膜上后，使用45Ca++配体印迹分析检测了大肠杆菌F0 ATP合酶质子通道的8 kDa亚基c的Ca++结合活性。纯化后的亚基c与45Ca++强烈结合，其Ca++结合特性与叶绿体类囊体膜的8 kDa CF0亚基III非常相似。N端甲酰甲硫氨酸羰基似乎对Ca++结合能力是必需的，通过用温和酸处理去除甲酰基后Ca++结合的丧失得以证明。二环己基碳二亚胺反应性的Asp - 61不参与Ca++结合，通过在两个大肠杆菌突变体Asp61→Asn和Asp61→Gly中Ca++结合得以保留证明。在大肠杆菌和类囊体的8 kDa蛋白质中，Ca++结合都依赖于pH，在pH 5.0时不存在，在pH 9.0附近达到最大值。一种预计会增加Ca++对其F0结合位点结合亲和力的处理（氯丙嗪光亲和附着）导致了全细胞中由碱到酸的pH跃变驱动的ATP形成受到抑制。当在氯丙嗪光亲和处理期间细胞中存在Ca++螯合剂EGTA时，未观察到抑制作用。在UV加氯丙嗪处理期间，使用EGTA - Ca++缓冲系统控制游离Ca++浓度，得到了对UV加氯丙嗪效应负责的位点上的表观Ca++结合常数接近80 - 100 nM。这些数据与这样的观点一致，即结合到大肠杆菌F0质子通道周质侧的Ca++可以阻止H+进入通道。在类囊体膜中也发生类似的效应，但Ca++结合位点在类囊体的腔侧，在那里Ca++结合可以调节酸碱跃变ATP形成。Ca++与F0和CF0复合物的结合与用于控制H+离子通过H+通道开口通量的pH依赖性门控机制一致。
