ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

我们的目标是了解阴离子交换在一个简单的（细菌） 系统 希望这将澄清小规模的 分子进出活细胞，这一基本知识 将深入了解分子事件， 调节正常的运输和代谢。 预计将有几种研究途径。 利用对这两个国家的研究， 完整的细胞和重建的系统，我们将检查革兰氏阴性， 革兰氏阳性细胞根据底物对阴离子交换进行分类 特异性、离子选择性和交换化学计量。 有利的 然后选择一个病例进行动力学研究，以区分乒乓球和 同步机制。 同时进行的工作将侧重于净化 来自乳酸链球菌的磷酸：糖6-磷酸反向转运蛋白。 这 是细菌阴离子交换的最佳表征实例。 它有一个 磷酸盐亲和力足够高，以允许结合试验，这应该 补充动力学研究的信息， 化学计量/选择性。 与此同时， 该反向转运蛋白的合成将被克隆到大肠杆菌中，使用 穿梭载体pSA 3。 具体目标是扩增蛋白质 内容物，作为纯化的辅助物，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 独立的研究将尝试直接测试运输， 在微粒体膜中发现的假定的糖磷酸载体， 天然和重组材料。 这与血糖控制有关 由肝脏生产。 进一步的工作将集中在ATP驱动的阳离子运输在S。乳酸。 作为 作为BFoF 1持续研究的一部分，我们将融合蛋白脂质体， 含有具有平面双层的BFoF 1，以测量宏观H+ ATP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.