ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

我们的目标是了解简单的(细菌)中的阴离子交换 系统。希望这将澄清小规模的 分子进出活细胞，而这一基本知识 将提供对分子事件的见解，这些事件有助于 调节正常的运输和代谢。 预计会有几种研究途径。利用对这两个问题的研究 完整的细胞和重组系统，我们将检查革兰氏阴性和 革兰氏阳性细胞根据底物对阴离子交换进行分类 专属性、离子选择性和交换化学计量学。一个有利的 然后将选择案例进行动力学研究，以区分乒乓球和 同时发生的机制。同时进行的工作将集中在提纯 磷酸：来自乳酸链球菌的糖6-磷酸逆向转运蛋白。这 是细菌阴离子交换最典型的例子。它有一个 磷酸盐亲和力高到足以进行结合测试，这应该 补充动力学研究中的信息和 化学计量学/选择性。同时基因(S)执导 该反向转运蛋白的合成将被克隆到大肠杆菌中，使用 航天飞机载体，pSA3。具体的目标是放大蛋白质 内容物，作为纯化的附件，并提供DNA序列 活性位点突变体中受影响的区域可以位于主要的 结构。独立研究将尝试通过一种 在微粒体膜中发现假定的糖磷酸盐载体，使用这两种 原生的和重组的材料。这与控制血糖有关 由肝脏产生。 其他工作将集中在乳酸链霉菌中由ATP驱动的阳离子转运。AS 作为BFoF1持续研究的一部分，我们将融合蛋白脂质体 含有平面双层膜的BFoF1，用于测量宏观H+ 由三磷酸腺苷水解产生的电流。

Our goal is an understanding of anion exchange in a simple (bacterial) system. It is hoped that this will clarify the mechanism by which small molecules enter and exit living cells, and that this fundamental knowledge will give insight into the molecular events which contribute to the regulation of normal transport and metabolism. Several avenues of research are anticipated. Using studies of both the intact cell and reconstituted systems, we will examine Gram-negative and Gram-positive cells to classify anion exchanges according to substrate specificity, ionic selectivity and exchange stoichiometry. A favorable case will then be chosen for kinetic studies to distinguish Ping Pong and Simultaneous mechanisms. Concurrent work will focus on purification of the phosphate: sugar 6-phosphate antiporter from Streptococcus lactis. This is the best characterized example of bacterial anion exchange. It has a phosphate affinity high enough to allow tests of binding, and this should complement the information from studies of kinetics and stoichiometry/selectivity. At the same time the gene(s) directing synthesis of this antiporter will be cloned into Escherichia coli, using the shuttle vector, pSA3. The specific goals are to amplify protein content, as an adjunct to purification, and to provide a DNA sequence so that regions affected in active site mutants can be located in the primary structure. Independent studies will attempt direct tests of transport by a putative sugar phosphate carrier found in microsomal membranes, using both native and reconstituted material. This relevant to control of glucose production by the liver. Additional work will focus on ATP-driven cation transport in S. lactis. As part of a continuing study of BFoF1, we will fuse proteoliposomes containing BFoF1 with planar bilayers, to measure the macroscopic H+ current generated on ATP hydrolysis.