Bishop L, Agbayani R, Ambudkar S V, Maloney P C, Ames G F
Department of Biochemistry, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1989 Sep;86(18):6953-7. doi: 10.1073/pnas.86.18.6953.
The histidine periplasmic permease of Salmonella typhimurium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. Proteoliposomes also transport histidine when GTP replaces ATP. Proteoliposomes do not catalyze significant ATP hydrolysis until histidine transport is initiated by addition of substrate along with HisJ, the water-soluble histidine-binding protein. Both initially and throughout the course of substrate transport there is a concomitant hydrolysis of ATP, with an apparent stoichiometry (ATP/histidine) of 5:1. These experiments demonstrate directly that ATP is the source of energy for periplasmic permeases, thus resolving previous controversies on this topic.
鼠伤寒沙门氏菌的组氨酸周质通透酶已被部分纯化并重组到蛋白脂质体中。在这种体外制备物中，转运活性完全依赖于所有四种通透酶蛋白（HisJ、HisQ、HisM和HisP）的存在以及内部ATP。重组系统显示出的初始转运速率与外翻膜囊泡获得的速率相当，并且它建立了100倍的组氨酸浓度梯度。当GTP取代ATP时，蛋白脂质体也能转运组氨酸。直到通过添加底物以及水溶性组氨酸结合蛋白HisJ启动组氨酸转运，蛋白脂质体才会催化显著的ATP水解。在底物转运的初始阶段和整个过程中，都伴随着ATP的水解，其明显的化学计量比（ATP/组氨酸）为5:1。这些实验直接证明ATP是周质通透酶的能量来源，从而解决了此前关于该主题的争议。
