MOLECULAR BIOLOGY OF BRUSH BORDER TRANSPORT PROTEINS

长期目标是阐明其结构和功能。 钠离子驱动的氨基酸共转运体。这些研究的关键是我最近 兔和人刷状边界Na+/葡萄糖基因克隆成功 共转运蛋白在非洲爪哇卵母细胞中的功能表达。具体的 目标如下： 1)克隆和测序肠道Na+/氨基酸转运体，如 利用表达克隆技术实现肠道Na+/Pro共转运蛋白 它是在克隆Na+/葡萄糖共转运蛋白的过程中进化出来的。克隆人 将被测序，推导出的一级结构将被用来 预测膜的二级结构和跨膜区域。 2)用生化和免疫化学方法挑战预测 技术，并通过现场定向分析转运蛋白的功能 突变体在非洲爪哇卵母细胞中的诱变和表达。抗体会 针对运输机的亲水性区域做好准备。西式 分析、蛋白质分解实验、抗体结合研究以刷边界 囊泡，糖基化实验将被用来探测次级 结构。Na+/Pro共转运蛋白的酪氨酸和赖氨酸残基 将被诱变以评估它们对Na+和Pro的可能影响 有约束力的。 3)研究与克隆氨基酸相关的遗传缺陷 用Western、Northern和Southern杂交分析共转运蛋白。

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.