STRUCTURE AND FUNCTION OF MEMBRANE TRANSPORT PROTEINS

膜运输蛋白的结构和功能

• 批准号: 3227623
• 负责人: DANIEL S LEVY
• 金额: $ 21.92万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-08-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227623
• 关键词: affinity labeling; anions; carbohydrate structure; chemical fingerprinting; complementary DNA; gene expression; ion transport; laboratory mouse; laboratory rabbit; laboratory rat; liposomes; liver cells; membrane activity; membrane channels; membrane proteins; membrane reconstitution /synthesis; membrane structure; messenger RNA; molecular cloning; monoclonal antibody; neoplastic cell culture for noncancer research; nucleic acid probes; phase change; photochemistry; protein sequence; protein structure; proteolysis; radiotracer; sulfates; tissue /cell culture; transport proteins

Organic anions such as the bile acids play a critical role in numerous physicological processes such as digestion of dietary lipids and regulation of cholesterol levels. These compounds are transported into the liver by a 49 kDa protein located in the sinusoidal plasma membrane. Defects in this transport system can result in intrahepatic cholestasis leading to numerous pathological conditions including liver injury and failure. The purpose of this research program is to investigate the structure of this transport system and its expression in different physiological and pathological states. The specific aims of the proposal are: 1) to isolate a cDNA clone for the Na+ -dependent anion transport protein. The amino acid sequence of this protein will be obtained by sequencing this cDNA clone; 2) to characterize the membrane organization as well as functionally important sites on this transport protein using a library of monoclonal antibodies, protein site specific and membrane domain specific labeling reagents and proteolytic dissection in conjunction with the sequences data obtained in (1); 3) to investigate the structure of a 49 kDa non- functioning transport protein in hepatoma cell plasma membranes; 4) to utilize the cDNA probe of the anion transport protein to characterize the steady state levels of mRNA for this protein in liver in different stages of development, during regeneration, in transformed hepatocytes and in other tissues; 5) to characterize the expression of this transport protein in the hepatocyte system described in (4) using mono and polyclonal antibodies against the transport protein; 6) to transfect the cDNA clone for this protein into non-transporting heptoma cells. These studies, which will provide important new information on the structure of the Na+ - dependent organic anion transport system and its expression in different physiological states, should ultimately lead to an understanding of the mechanism of action and regulation of this membrane system.

胆汁酸等有机阴离子在 许多生理过程，例如食物的消化 脂质和胆固醇水平的调节。 这些化合物是 通过位于 49 kDa 的蛋白质转运到肝脏 正弦质膜。 该运输系统的缺陷 可导致肝内胆汁淤积，导致多种 病理状况包括肝损伤和衰竭。 这 该研究计划的目的是调查结构 该转运系统及其在不同生理状态下的表达 和病理状态。 该提案的具体目标是：1） 分离用于 Na+ 依赖性阴离子运输的 cDNA 克隆 蛋白质。 将获得该蛋白质的氨基酸序列 通过对该 cDNA 克隆进行测序； 2）表征膜 组织以及功能上重要的网站 使用单克隆抗体库运输蛋白质、蛋白质 位点特异性和膜域特异性标记试剂和 结合序列数据进行蛋白水解解剖 在(1)中获得； 3）研究49 kDa非-的结构 肝癌细胞质膜中的功能转运蛋白； 4)利用阴离子转运蛋白的cDNA探针 表征该蛋白质的 mRNA 稳态水平 肝脏处于不同的发育阶段、再生过程、 转化的肝细胞和其他组织； 5）表征 该转运蛋白在肝细胞系统中的表达 (4)中描述的使用单克隆和多克隆抗体 转运蛋白； 6) 转染该蛋白的cDNA克隆 进入非运输性肝癌细胞。 这些研究将 提供有关 Na+ 结构的重要新信息 - 依赖的有机阴离子转运系统及其在体内的表达 不同的生理状态，最终会导致 了解其作用机制和调节 膜系统。