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Molecular mechanism of energy coupling in active transport

主动运输中能量耦合的分子机制

基本信息

批准号：
06044153
负责人：
TSUCHIYA Tomofusa
金额：
$ 5.63万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044153/
关键词：
Active transport Energy coupling Symporter Antiporter Functional domain

项目摘要

Molecular mechanism of energy coupling in active transport of solutes across biological membranes was studied from biochemical and genetic points of view.First we analyzed the melibiose transport system of Escherichia coli, Salmonella typhimurium and Enterobacter aerogenes, the glucose transport system of Vibrio parahaemolyticus, and so on. We characterized these transport systems, and energy coupling in these systems were revealed. We constructed chimeric transport proteins from, for example, the melibiose transporter of E.coli and that of S.typhimurium Although the melibiose transporters of these two organisms are highly homologous in the primary structure, they showed different cation coupling. Analysis of the chimeric proteins revealed domains imvolved in the cation coupling, The glucose transporter of V.parahaemolyticus (Na+/glucose symporter) showed similarity in the primary structure with human SGLT (Na+/glucose symporter). Thus, several amino acid residues which seem to be important for cation recogonition or substrate (or inhibitor) recognition have been intentified.The sugar phosphate/inorganic phosphate antiporter of E.coli has been overexpressed and purified using several genetic techniques. The biochemical properties and energy coupling in the antiporter was characterized. The techniques utilized in the studies were revealed to be very useful in the study of membrane proteins, in general.Energy coupling in the Na+/H+ antiporters in E.coli, V.parahaemolyticus and Pseudomonas aeruginosa has been also investigated. We found that several amino acid residues in the Na+/H+ antiporter of E.coli are important for pH sensing or its activity. Membrane topology of the antiporter was revealed.Collaboration and discussion with foreign Scientists were very valuable.

从生物化学和遗传学的角度研究了溶质跨膜主动转运过程中能量耦合的分子机制，首先分析了大肠杆菌、鼠伤寒沙门氏菌和产气肠杆菌的蜜二糖转运系统，副溶血弧菌的葡萄糖转运系统等，并对这些转运系统进行了表征，揭示了这些转运系统中的能量耦合。我们从例如大肠杆菌的蜜二糖转运蛋白和鼠伤寒沙门氏菌的蜜二糖转运蛋白构建了嵌合转运蛋白。虽然这两种生物的蜜二糖转运蛋白在一级结构上高度同源，但它们显示出不同的阳离子偶联。对嵌合蛋白的结构分析表明，该蛋白具有参与阳离子偶联的结构域。副溶血性弧菌葡萄糖转运蛋白（Na+/glucose symporter）与人SGLT（Na+/glucose symporter）的一级结构相似。因此，几个氨基酸残基，似乎是重要的阳离子代谢或底物（或抑制剂）识别已加强。糖磷酸/无机磷酸盐反向转运蛋白的大肠杆菌已过量表达和纯化，利用几种遗传技术。对反向转运蛋白的生化性质和能量耦合进行了表征。研究中所采用的技术在膜蛋白的研究中是非常有用的，在一般情况下。能量耦合在大肠杆菌，副溶血性弧菌和铜绿假单胞菌的Na ~+/H ~+反向转运蛋白也进行了研究。我们发现大肠杆菌Na+/H+逆向转运蛋白中的几个氨基酸残基对于pH传感或其活性是重要的。揭示了反向转运蛋白的膜拓扑结构，并与国外学者进行了有益的合作和探讨。