MEMBRANES AND ACTIVE TRANSPORT OF AMINO ACIDS

膜和氨基酸的主动运输

基本信息

批准号：
6342424
负责人：
GIOVANNA F AMES
金额：
$ 49.75万
依托单位：
CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-01-01 至 2002-12-31
项目状态：
已结题

项目摘要

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.

转运蛋白超家族、交通ATP酶（或ABC蛋白）、囊性纤维化跨膜电导调节因子（CFTR），P- 多药耐药性（MDR）糖蛋白和细菌周质渗透。多重耐药性是癌症的主要问题之一化疗囊性纤维化是白种人最常见的隐性遗传病疾病。周质通透酶已被广泛研究并提供一个很好的模型系统，用于理解作用机制超家族的医学相关真核成员。这样的一位通透酶（组氨酸通透酶）已被详细表征。按原样一般来说，对于交通 ATP 酶来说，组氨酸通透酶由以下组成：两个疏水域是膜的组成部分，以及两个疏水域亲水结构域也插入膜中并结合 ATP。 ATP的水解被用作能量来源。由于 CFTR 看起来是一个通道，因此确定是否原核系统也起到通道的作用。这将是一个完全原核系统的新概念。从已知的结构膜结合复合物，疏水域确实有可能周质渗透酶形成底物穿过的通道膜上，ATP 水解产生必要的构象变化。周质通透酶的一个特有特征是存在将底物集中在外部的受体膜结合复合物的表面。受体发送信号给膜结合复合物，导致 ATP 水解和易位。本研究中将使用的工具包括几个重构的工具系统和几种可测量的酶活性，允许在体外功能测定。作为通道的交通ATP酶的活性将是在脂质双层中进行了研究。之间的信号传输机制可溶性受体和膜结合复合物，特别是能量- 耦合成分，将通过生化反应进行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，并通过遗传分析分离具有改变的信号传导过程的突变体。相似的生化和遗传程序将用于研究结构膜结合复合物。此外，该组件的膜结合复合物将被单独纯化和表征。将尝试二维和三维晶体学了解复合体和亚基的结构。除了解决与作用机制相关的基本问题一般来说，对这种原核模型系统的研究将帮助真核研究人员努力解决这个问题与多重耐药性、囊性纤维化、疟疾相关的医疗问题寄生虫遏制等。