MEMBRANES AND ACTIVE TRANSPORT OF AMINO ACIDS

膜和氨基酸的主动运输

• 批准号: 3568390
• 负责人: GIOVANNA F AMES
• 金额: $ 37.64万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3568390
• 关键词: Salmonella typhimurium; X ray crystallography; active transport; adenosinetriphosphatase; aminoacid transport; biological signal transduction; covalent bond; enzyme mechanism; genetic manipulation; histidine; lipid bilayer membrane; membrane channels; membrane permeability; membrane proteins; membrane reconstitution /synthesis; membrane structure; microorganism metabolism; mutant; permease; protein structure; proteolysis; receptor coupling; Salmonella typhimurium; X ray crystallography; active transport; adenosinetriphosphatase; aminoacid transport; biological signal transduction; covalent bond; enzyme mechanism; genetic manipulation; histidine; lipid bilayer membrane; membrane channels; membrane permeability; membrane proteins; membrane reconstitution /synthesis; membrane structure; microorganism metabolism; mutant; permease; protein structure; proteolysis; receptor coupling

The superfamily of translocators, traffic ATPases (or ABC proteins), the cystic fibrosis transmembrane conductance regulator (CFTR), the P- glycoprotein of multidrug resistance (MDR), and bacterial periplasmic permeases. Multidrug resistance is one of the major problems in cancer chemotherapy an cystic fibrosis is the most common recessive caucasian disease. Periplasmic permeases have been extensively studied and provide a good model system for understanding the mechanism of action of the medically relevant eukaryotic members of the superfamily. One such permease, the histidine permease, has been characterized in detail. As is true for traffic ATPases in general, the histidine permease is composed of two hydrophobic domains that are integral parts of the membrane, and of two hydrophilic domains that are also inserted into the membrane and bind ATP. Hydrolysis of ATP is used as the energy source. Since CFTR appears to be a channel, it is important to determine whether prokaryotic systems also function as channels. This would be an entirely novel concept for the prokaryotic systems. From the known structure of the membrane-bound complex, it is indeed possible that the hydrophobic domains of periplasmic permeases form a channel through which the substrate crosses the membrane, with ATP hydrolysis resulting in the necessary conformational changes. A characteristic peculiar to periplasmic permeases is the presence of a receptor that concentrates the substrate at the external surface of the membrane-bound complex. The receptor sends a signal to the membrane-bound complex, resulting in ATP hydrolysis and translocation. Among the tools that will be used in this study are several reconstituted systems and several measurable enzymatic activities that permit in vitro assays of function. The activity of traffic ATPases as channels will be investigated in lipid bilayers. The mechanism of signaling between the soluble receptor and the membrane-bound complex, in particular the energy- coupling component, will be studied by the use of biochemical reactions that distinguish between different conformations of proteins, such as limited proteolysis and covalent labeling, and by genetic analysis through the isolation of mutants with altered signaling processes. Similar biochemical and genetic procedures will be used to study the architecture of the membrane-bound complex. In addition, the components of the membrane-bound complex will be purified and characterized individually. Both two- and three-dimensional crystallography will be attempted to understand the structures of both the complex and the subunits. In addition to solving basic questions related to the mechanism of action of permeases in general, the study of this prokaryotic model system will help the efforts of eukaryotic researchers towards a solution of the medical problems related to multidrug resistance, cystic fibrosis, malarial parasite containment, and others.

易位者的超家族，流量ATPases（或ABC蛋白）， 囊性纤维化跨膜电导调节剂（CFTR），P- 多药耐药性（MDR）和细菌周质的糖蛋白 腐蚀。 多药抵抗是癌症的主要问题之一 化学疗法囊性纤维化是最常见的隐性高加索人 疾病。 周质腐蚀已被广泛研究并提供 一个良好的模型系统，用于理解行动机理 超家族与医学相关的真核生物成员。 一个这样的 渗透酶是组氨酸渗透酶，已详细表征。 原来 通常，对于流量ATPases，组氨酸渗透酶由 两个是膜的组成部分的两个疏水结构域，两个 也插入膜并结合ATP的亲水结构域。 ATP的水解用作能源。 由于CFTR似乎是一个渠道，因此必须确定是否是否 核系统也充当通道。 这完全是 核系统的新颖概念。 来自已知的结构 结合膜的复合物，确实有可能疏水结构域 底质腐蚀的形成一个通道，底物通过该通道越过 膜进行ATP水解，导致必要的构象 更改。 周质腐蚀的特征是 存在将底物浓缩在外部的受体 膜结合复合物的表面。 受体向 膜结合的复合物，导致ATP水解和易位。 在本研究中将使用的工具中有几个重构 系统和几种可测量的酶活性 功能测定。 流量ATPases的活动作为渠道 在脂质双层中进行了研究。 在 可溶性受体和结合膜的复合物，特别是能量 耦合组件将通过使用生化反应来研究 区分蛋白质的不同构象，例如 有限的蛋白水解和共价标记，以及通过遗传分析 分离有改变信号过程的突变体。 相似的 生化和遗传程序将用于研究体系结构 膜结合的复合物。 另外， 膜结合的复合物将被纯化并单独表征。 二维和三维晶体学都将尝试 了解复合物和亚基的结构。 除了解决与行动机制有关的基本问题 通常，该核模型系统的研究将 帮助真核研究人员努力解决方案 与多药耐药性，囊性纤维化，疟疾有关的医学问题 寄生虫遏制等。