Kim Y J, Rhee S K
Department of Microbiology, Changwon National University, Korea.
Mol Cells. 1997 Aug 31;7(4):473-7.
We studied the effect of a H+ electrochemical potential generated by F1F0 ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F1F0 ATPase of V. alginolyticus catalyzed the pumping of H+ coupled to ATP hydrolysis as measured in fluorescence quenching experiments. Consequently, this enzyme leads to the generation of a H+ electrochemical potential. The H+ electrochemical potential generated by F1F0 ATPase was completely abolished by 30 microM N,N'-dicyclohexylcarbodiimide (DCCD) or 5 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30 degrees C. The treatment of membrane vesicles with 30 microM DCCD at 30 degrees C had little influence on the translocation activity of uncleavable OmpF-Lpp, a model secretory protein, as compared to the intact membrane vesicles. On the other hand, the NADH:quinone oxidoreductase of V. alginolyticus is known to be a Na+ pump that leads to generation of a Na+ electrochemical potential. This Na+ electrochemical potential stimulates protein translocation into inside-out membrane vesicles prepared from V. alginolyticus in the presence of Escherichia coli SecA [Tokuda, H., Kim, Y. J., and Mizushima, S. (1990) FEBS Lett. 264, 10-12] From these results, it is evident that the stimulatory effect of the Na+ electrochemical potential on protein translocation in V. alginolyticus is not affected by the H+ electrochemical potential influence of F1F0 ATPase.
我们研究了F1F0 ATP酶产生的H⁺电化学势对模型蛋白体外转运至溶藻弧菌内翻膜囊泡的影响。如荧光猝灭实验所测，溶藻弧菌的F1F0 ATP酶催化与ATP水解偶联的H⁺泵出。因此，这种酶导致H⁺电化学势的产生。在30℃时，30微摩尔N，N'-二环己基碳二亚胺（DCCD）或5微摩尔羰基氰化物间氯苯腙（CCCP）可完全消除F1F0 ATP酶产生的H⁺电化学势。与完整膜囊泡相比，在30℃用30微摩尔DCCD处理膜囊泡对不可切割的OmpF-Lpp（一种模型分泌蛋白）的转运活性影响很小。另一方面，已知溶藻弧菌的NADH：醌氧化还原酶是一种Na⁺泵，可导致Na⁺电化学势的产生。在大肠杆菌SecA存在的情况下，这种Na⁺电化学势刺激蛋白转运至由溶藻弧菌制备的内翻膜囊泡中[德田浩、金英姬和水岛淑（1990年）《欧洲生物化学学会联合会快报》264，10 - 12]。从这些结果可以明显看出，Na⁺电化学势对溶藻弧菌中蛋白转运的刺激作用不受F1F0 ATP酶的H⁺电化学势影响。
