MEMBRANES AND ACTIVE TRANSPORT OF AMINO ACIDS

该项目涉及确定物质被吸收的机制。 运输进出细胞，并分析生物合成， 细菌膜的结构。 这些实验将利用 微生物（鼠伤寒沙门氏菌），可以通过基因操作 分离在主动转运的各个步骤中有缺陷的突变体， 膜生物发生，然后可以进行生物化学分析。 组氨酸 交通运输系统，我们使用作为一个模型系统，是由三个 蛋白质：组氨酸结合蛋白J（周质，已表征）， P蛋白（膜结合）和Q蛋白。 P蛋白将是 纯化和表征，这是理解 运输的分子机制。 Q蛋白将被鉴定， 纯化和表征。 为了帮助我们，我们克隆了 组氨酸转运操纵子，并将能够识别缺失的组件， 产生大量蛋白质用于纯化目的。 克隆人还将 用于对这些基因的DNA进行测序，以了解它们的 调节，膜和周质蛋白的分泌机制。 我们 将试图在脂质体中重建主动转运系统， 原生质球 我们将研究周质蛋白与 膜的交联实验。 我们还开始研究 能量耦合机制在我们的系统中发挥作用：我们基本上计划 验证乙酰磷酸是周质的能量来源的假设 系统（Hong），通过分离乙酰磷酸盐生物降解中的适当突变体， 并直接测定细胞中乙酰磷酸的水平。 的 膜结合组分P蛋白的功能多样性将是 通过研究其与赖氨酸，精氨酸， 在体内和体外的鸟氨酸结合蛋白。

This project is concerned with defining mechanisms by which substances are transported into and out of cells and with analyzing the biosynthesis and structure of bacterial membranes. The experiments will make use of microorganisms (Salmonella typhimurium) which can be manipulated genetically by isolating mutants defective in the various steps of active transport or of membrane biogenesis, which can then be analyzed biochemically. The histidine transport system, which we use as a modely system, is composed of three proteins: the histidine-binding protein J (periplasmic, already characterized), the P protein (membrane bound) and the Q protein. The P protein will be purified and characterized, which is an essential step in the understanding of the molecular mechanism of transport. The Q protein will be identified, purified, and characterized. To aid us in these efforts, we have cloned the histidine transport operon and will be able to identify missing components and produce large amounts of protein for purification purposes. The clone will also be used for sequencing the DNA of these genes in order to understand their regulation, the secretion mechanism of membrane and periplasmic proteins. We will attempt to reconstitute an active transport system in liposomes or spheroplasts. We will research the interaction of periplasmic proteins with membranes by cross-linking experiments. We are also starting a study of the energy coupling mechanisms functioning in our system: we essentially plan to test the hypothesis that acetylphosphate is the energy source for periplasmic systems (Hong) by isolating appropriate mutants in acetylphosphate biosynthsis and by assaying directly the level of acetylphosphate in the cell. The multiplicity of function of the membrane-bound component, P protein, will be investigated by studying its involvement with a lysine-, arginine-, ornithine-binding protein in vivo and in vitro.