ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

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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.