MOLECULAR ARCHITECTURE OF THE NA/GLUCOSE COTRANSPORTER

本提案的长期目标是确定Na+/葡萄糖 协同转运蛋白实际上是跨膜转运Na+和葡萄糖。 到 为了实现这一目标，有必要解开分子结构， 转运蛋白 短期目标是确定Na+和 糖结合位点的蛋白质和长期目标是划定 分离蛋白的结构拓扑和构象。 协同转运蛋白上的Na+、糖和抑制剂（根皮苷）结合位点 将使用三种策略进行识别：第一，是标记 酪氨酸修饰刷状缘膜囊泡的Na+/葡萄糖共转运 存在于Na+结合位点的残基和存在于Na+结合位点的赖氨酸残基 葡萄糖和根皮苷结合位点。 蛋白质将被切割， 蛋白水解，纯化和测序;第二，是采用分子生物学技术， 突变酪氨酸和赖氨酸残基的工程技术被认为是 在活跃的网站。 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/或赖氨酸残基在Na+/葡萄糖中起关键作用 共转运;第三，我们将使用分子技术来测试 Gly 43和Arg 300参与Na+结合和转运。 将在非洲爪蟾卵母细胞中测定突变体的转运特性， 免疫沉淀技术将用于确认 卵母细胞质膜中的突变蛋白。 我们最初的战略 以实现纯化Na+/葡萄糖的长期目标 协同转运蛋白将涉及在E.在培养的昆虫中 细胞 融合蛋白将在细菌和杆状病毒中表达 构建体将用于在SF 9细胞中过表达协同转运蛋白。 在高表达水平的情况下，至少两个数量级大于 在肠上皮细胞中，我们将很好地定位于纯化蛋白质， 电泳和色谱法。 这种蛋白质将首先用于 提高抗体，但长期目标是使用蛋白质， 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.