MOLECULAR ARCHITECTURE OF THE NA/GLUCOSE COTRANSPORTER

这项提议的长期目标是确定Na+/葡萄糖如何 辅转运蛋白实际上是跨膜转运Na+和葡萄糖的。至 为了达到这一目的，有必要解开分子的结构 运输蛋白。短期目标是确定Na+和 蛋白质的糖结合部位和长期目标是描绘 分离蛋白的结构拓扑和构象。 共转运蛋白上的Na+、糖和抑制物(根茎苷)结合部位 将使用三种策略来识别：第一，是将 酪氨酸作用下刷状缘膜囊泡的Na+/葡萄糖共转运 发生在Na+结合部位的残基和位于 葡萄糖和连翘苷的结合部位。这种蛋白质会被 蛋白质分解，纯化和测序；第二，是使用分子 突变酪氨酸和赖氨酸残基的工程技术被认为是 在活跃的地点。然后我们将确定这些是否是 用组特异性试剂标记残留物，并确定是否 酪氨酸和/或赖氨酸残基与Na+/葡萄糖密切相关 第三，我们将使用分子技术来测试 假设Gly43和Arg300参与Na+结合和转运。 突变体的转运特性将在非洲爪哇卵母细胞中进行检测，并 将利用免疫沉淀技术来确认是否存在 卵母细胞质膜中的突变蛋白。我们最初的战略 朝着净化Na+/葡萄糖的长远目标迈进 共转运蛋白将在大肠杆菌和培养的昆虫中过表达 细胞。融合蛋白将在细菌和杆状病毒中表达 构建物将被用来在sf9细胞中过表达共转运蛋白。 高表达水平，至少比 在肠道细胞中，我们将很好地通过以下方式纯化蛋白质 电泳法和层析法。这种蛋白质将首先用于 提高抗体，但长期目标是将这种蛋白质用于 结构和功能研究。

The long-term aim of this proposal is to determine how the Na+/glucose cotransporter actually transports Na+ and glucose across the membrane. To achieve this aim it is necessary to unravel the molecular architecture of the transport protein. The short-term goal is to identify the Na+ and sugar binding sites of the protein and the long-term goal is to delineate the structural topology and conformations of the isolated protein. The Na+, sugar and inhibitor (phlorizin) binding sites on the cotransporter will be identified using three strategies: The first, is to label the Na+/glucose cotransport in brush border membrane vesicles at tyrosine residues occurring at the Na+ binding site, and lysine residues at the glucose and phlorizin binding sites. The protein will be cleaved by proteolysis, purified, and sequenced; The second, is to employ molecular engineering techniques to mutate tyrosine and lysine residues thought to be at the active sites. We will then determine whether or not these are the residues labeled by group specific reagents, and establish whether or not the tyrosine and/or lysine residues are critically involved in Na+/glucose cotransport; and third, we will use molecular techniques to test the hypothesis that Gly43 and Arg300 are involved in Na+ binding and transport. Transport properties of mutants will be assayed in Xenopus oocytes, and immunoprecipitation techniques will be utilized to confirm the presence of the mutant proteins in the oocyte plasma membrane. Our initial strategy toward achieving the long-term goal of purifying the Na+/glucose cotransporter will involve overexpression in E. coli and in cultured insect cells. Fusion proteins will be expressed in bacteria, and baculovirus constructs will be used to overexpress the cotransporter in sf9 cells. With high expression levels, at least two orders of magnitude greater than in enterocytes, we will be well positioned to purify the protein by electrophoresis and chromatography. The protein will first be used to raise antibodies, but the long-term objective is to use the protein for structure and functional studies.