CHARACTERIZATION OF THE LYSOSOMAL ANION TRANSPORTER

溶酶体阴离子转运蛋白的表征

• 批准号: 3245746
• 负责人: ADAM J JONAS
• 金额: $ 16.46万
• 依托单位: LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245746
• 关键词: acidity /alkalinity; anions; antiport; band 3 protein; bicarbonates; bromine; fluorine; genetic library; human tissue; inhibitor /antagonist; iodine; ion transport; kidney cell; laboratory rabbit; laboratory rat; liver cells; lysosomes; membrane potentials; membrane transport proteins; metabolism; molecular cloning; protein structure function; selenium; stilbenes; sulfates; sulfonate; taurine; temperature; thiocyanates; tissue /cell culture; transport proteins; western blottings

This proposal is designed to characterize the function and structure of the lysosomal anion (sulfate) transporter, a newly recognized member of a family of proteins that are critical to cell function. These investigations will provide insights into basic lysosomal function and will form a framework for the study of this and other clinically significant lysosomal transport proteins. A considerable amount of biochemical, immunological and molecular information is available concerning the functionally homologous plasma membrane anion transporters. This information will now be applied toward study of the lysosomal system according to the following specific aims. 1) Define the substrated of lysosomal anion transport: Sulfate, chloride, and molybdate have already been identified as substrates for transport. Additional divalent anions such as SeO4-2 and monovalent anions such as HCO33-, F-, Br-, I-_, and SCN-, will be examined. Effects of pH, membrane potential and temperature will be determined. Affinity constants and rates of transport will be calculated. Emphasis will be placed further defining the role of protons as co- transported species. 2) Define the role of lysosomal anion transport in cell metabolism: Transport will be examined in lysosomes form other tissues, including kidney. Using cultured cell systems, the role of the lysosomes in regulating intracellular anion concentrations through recycling will be examined. Whether in vitro substrates for transport such as molybdate are also in vivo substrates will be determined. The role of the transporter in the regulation of lysosomal pH and chloride movement will also be examined. 3) Identify inhibitors of lysosomal anion transport: Inhibitors of the erythrocyte band 3 anion transporter including stilbene sulfonic acids, NAP-taurine, and arylisothiocyanates will be screened for their effect on the lysosomal system in order to provide information on structure and function. One important goal of these studies is to identify irreversible inhibitors that can subsequently be used as probes to aid in identification of the lysosomal transport protein. 4) Identification of the lysosomal anion transport protein Strategies for identification of the lysosomal transporter include: 1) Use of irreversible inhibitors of lysosomal membrane anion transport as probes, 2) Use of immunoblots to evaluate antibodies against the highly conserved carboxyl ends of the AE1 (BAND 3) and AE3 anion transporters for evidence of cross reactivity with the lysosomal protein, and 3) Screening rat liver cDNA libraries for hybridization with the 3' portion of a mouse AE1 cDNA probe under conditions of reduced stringency. Success with any of these techniques will be followed by attempts to clone the gene for the lysosomal transporter.

该建议旨在表征 溶酶体阴离子（硫酸盐）转运蛋白，一个新认识的成员 对细胞功能至关重要的蛋白质家族。 这些 调查将为基本溶酶体功能和 将构成研究和其他临床研究的框架 明显的溶酶体转运蛋白。 大量 提供生化，免疫学和分子信息 关于功能上同源的质膜阴离子转运蛋白。 现在，此信息将用于研究溶酶体系统 根据以下特定目的。 1）定义溶酶体阴离子转运的底座： 硫酸盐，氯化物和钼酸盐已经被鉴定为 运输的基材。 其他二价阴离子，例如SEO4-2和 HCO33-，F-，BR-，I-_和SCN-等单价阴离子将是 检查。 pH，膜电位和温度的影响 决定。 将计算亲和力常数和运输速率。 重点将进一步定义质子的作用 运输的物种。 2）定义溶酶体阴离子转运在细胞代谢中的作用： 将在溶酶体中检查运输，包括其他组织 肾。 使用培养的细胞系统，溶酶体在 通过回收调节细胞内阴离子浓度将是 检查。 在体外的运输底物（例如钼酸盐）是否为 还将确定体内底物。 转运蛋白的作用 在调节溶酶体pH和氯化物运动中也将是 检查。 3）确定溶酶体阴离子转运的抑制剂： 红细胞带3阴离子转运蛋白的抑制剂，包括stilbene 将筛选磺酸，nap-taurine和芳基硫氰酸盐的 它们对溶酶体系统的影响，以提供有关 结构和功能。 这些研究的一个重要目标是 识别不可逆的抑制剂，随后可以用作探针 帮助鉴定溶酶体转运蛋白。 4）鉴定溶酶体阴离子转运蛋白 鉴定溶酶体转运蛋白的策略包括：1）使用 溶酶体膜阴离子转运的不可逆抑制剂的抑制剂 探针，2）使用免疫印迹评估对高度的抗体 AE1的保守羧基（频段3）和AE3阴离子转运蛋白 与溶酶体蛋白交叉反应性的证据，3）筛选 大鼠肝cDNA文库与小鼠的3'部分杂交 在严格降低的条件下，AE1 cDNA探针。 成功 在这些技术之后，将尝试克隆基因 溶酶体转运蛋白。