STRUCTURE AND FUNCTIONS OF NA/GLUCOSE COTRANSPORTERS

NA/葡萄糖协同转运蛋白的结构和功能

• 批准号: 3235172
• 负责人: ERNEST M WRIGHT
• 金额: $ 18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-05-01 至 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235172
• 关键词: affinity chromatography; biological models; brush border membrane; carbohydrate structure; chemical binding; conformation; covalent bond; fluorescence polarization; fluorescent dye /probe; glucose transport; high performance liquid chromatography; laboratory rabbit; liposomes; monoclonal antibody; protein sequence; protein structure; small intestines; stop flow technique; transport proteins

It has long been established that the "active transport" of sugars, amino acids, and other metabolites is due to the presence of Na gradients across plasma membranes and Na-cotransport systems in the membranes. Recently, the intestinal brush border Na/glucosecotransporter has been identified using fluorescent covalent probes bound to the glucose and/or Na-active sites. Our long term goal is to determine the structure of the Na/glucose cotransporter and to correlate this structure with the transport properties of the polypeptide(s). The specific aims are: 1) isolate and purify the functional cotransporter to homogeneity. This will be accomplished by fluorescently labeling the glucose carrier covalently at the glucose and/or Na-binding sites, and following the enrichment of the protein through chromatofocusing and affinity chromatography; 2) determine the structure of the purified carrier (sugar content, amino acid composition, and amino acid sequences of the Na and glucose active sites); 3) determine the molecular topography of the active sites using fluorescent probes (fluorescent quenching, energy transfer, and native trypophan fluorescence); and 4) develop a structural and kinetic model of Na/glucose cotransport. Transport activity in highly purified brush border vesicles, soluble protein, and reconstituted systems will be measured using Na-dependent a) glucose transport, b) phlorizin binding, c) potential changes, and d) quenching of fluorescent probes on the glucose-site. These studies will have general impact on our understanding of the mechanism of sugar, amino acid, metabolic intermediate transport across plasma membranes of the intestine, liver, kidney and brain, and particular relevance to the absorption of sugars by the small intestine.

长期以来已经确定 糖，氨基酸和其他代谢产物是由于 跨质膜和 膜中的NA-跨转运系统。 最近， 肠道边界Na/glucosecotransporter已经 使用荧光共价探针确定 葡萄糖和/或NA活性位点。 我们的长期目标是 确定Na/葡萄糖共转运蛋白的结构和至 将这种结构与运输特性相关联 多肽。 具体目的是：1）隔离并净化功能 共转运蛋白到同质性。 这将通过 荧光将葡萄糖载体共价标记 葡萄糖和/或NA结合位点，然后遵循富集 通过色素和亲和力的蛋白质 色谱； 2）确定纯化的结构 载体（糖含量，氨基酸成分和氨基酸 Na和葡萄糖活性位点的序列）； 3）确定 使用荧光的活性位点的分子形貌 探针（荧光淬灭，能量转移和天然 锥虫荧光）； 4）发展结构和 Na/葡萄糖共转运的动力学模型。 高度纯化的刷子边框囊泡中的运输活动， 将测量可溶性蛋白质和重构系统 使用na依赖性a）葡萄糖转运，b）硫曲蛋白结合， c）潜在的变化，d）荧光探针在 葡萄糖位点。 这些研究将对我们对 糖，氨基酸，代谢中间体的机制 横跨肠道，肝脏的质膜， 肾脏和大脑，以及与吸收特别相关 小肠的糖。