MOLECULAR BIOLOGY OF BRUSH BORDER TRANSPORT PROTEINS

长期目标是阐明的结构和功能， Na+驱动的氨基酸协同转运蛋白。这些研究的关键是我最近 成功克隆兔和人刷状缘Na+/葡萄糖 在非洲爪蟾卵母细胞中的功能性表达。具体 目标如下： 1)为了克隆和测序肠道Na+/氨基酸转运蛋白， 肠Na+/脯氨酸共转运蛋白的表达克隆 其在Na+/葡萄糖协同转运蛋白的克隆过程中进化。克隆 将被测序，推导出的一级结构将用于 预测二级结构和跨膜区域。 2)为了挑战使用生物化学和免疫化学 技术，并通过现场定向 诱变和突变体在非洲爪蟾卵母细胞中的表达。抗体将 针对转运蛋白的亲水区域制备。西方 分析，蛋白水解实验，抗体结合研究，以刷状缘 囊泡和糖基化实验将被用来探测二级 结构. Na+/脯氨酸共转运蛋白的酪氨酸和赖氨酸残基 将进行诱变处理，以评估其对Na+和脯氨酸的可能影响 约束力 3)研究与克隆氨基酸相关的遗传缺陷 使用Western、北方和南方印迹分析共转运蛋白。

The long-term objective is the elucidation of the structure and function of Na+-driven amino acid cotransporters. The key to these studies is my recent success in cloning the rabbit and human brush border Na+/glucose cotransporter with functional expression in Xenopus oocytes. The specific aims are as follows: 1) To clone and sequence intestinal Na+/amino acid transporters such as the intestinal Na+/proline cotransportel,using the expression cloning technique which evolved during the cloning of the Na+/glucose cotransporter. Clones will be sequenced and the deduced primary structures will be used to predict the secondary structures and membrane-spanning regions. 2) To challenge the predictions using biochemical and immunochemical techniques and to analyze the function of the transporters by site-directed mutagenesis and expression of mutants in Xenopus oocytes. Antibodies will be prepared against hydrophilic regions of the transporters. Western analysis, proteolytic experiments, antibody binding studies to brush border vesicles, and glycosylation experiments will be used to probe the secondary structures. Tyrosine and lysine residues of the Na+/proline cotransporter will be mutagenized to assess their possible effects on Na+ and proline binding. 3) To study inherited defects associated with the cloned amino acid cotransporters using Western, Northern and Southern blot analysis.