BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION EXCHANGE

描述：本项目的长期目标是了解 跨细胞膜偶联转运的分子机制。 重点 该项目的重点是红细胞带3蛋白（也称为AE 1）， 它是红细胞膜的主要成分， 耦合输运机理研究的良好表征模型系统。 除了作为模型系统的价值外，带3介导的氯化物-硫酸盐 交流（本项目的主要重点）具有潜在的相关性， 研究了由β-淀粉样蛋白介导的硫酸盐转运（在 腺瘤）蛋白和负责变形性腺瘤的蛋白， 软骨形成的遗传性疾病。 一般的做法是采取 带3的丰度对研究结构-功能关系的优势 通过生物化学方法，并比较生物化学研究的结果 与通过定点诱变获得的那些相同。 第一个目标涉及一个 特别是谷氨酸残基（人E681;小鼠E699），其在 阴离子移位事件。 工作假设，基于化学 改性实验，是在这个残基上的负电荷， 通常沿着沿着大部分跨膜电场移动 氯化物和两个蛋白质结合的正电荷，导致 电中性易位事件。 这个想法将在几个测试 方法，通过使用化学修饰的人类带3和转基因小鼠 表达在该残基处突变的条带3。 使用的方法包括 底物结合测量，预稳态和稳态示踪剂 通量测量和膜片钳电记录。 第二个目标是 使用改进的色谱方法分析疏水肽 以赖氨酸的位置为基础，完成第3带的全拓扑图 残基 这些研究应该解决目前关于乐队的争议 3拓扑学。 第三个目标是使用已公布的方法来表达频带3 酵母（酿酒酵母）中的膜结构域，用于 定点突变的功能分析。 第四个目标是 使用生物化学方法和诱变鉴定第二个羧基， 与转运途径相关的组（E681除外）， 定位外表面赖氨酸残基参与亚基接触。 的 诱变将在两个非洲爪蟾卵母细胞中表达的条带3中进行 和酵母中。

DESCRIPTION: The long-term goal of this project is to understand the molecular mechanism of coupled transport across cell membranes. The focus of the project is on the red blood cell band 3 protein (also known as AE1), which is a major component of the red cell membrane and is a well-characterized model system fo mechanistic studies of coupled transport. In addition to its value as a model system, band 3-mediated chloride-sulfate exchange (the main emphasis of this project) is of potential relevance to the study of sulfate transport mediated by the DRA (down-regulated in adenoma) protein and the protein responsible for diastrophic displasia, an inherited disorder of cartilage formation. The general approach is to take advantage of the abundance of band 3 to study structure-function relations by biochemical methods, and to compare the result of biochemical studies with those obtained by site-directed mutagenesis. The first aim concerns a particular glutamate residue (human E681; mouse E699), which has a role in the anion translocation event. The working hypothesis, based on chemical modification experiments, is that the negative charge on thi residue normally moves across much of the transmembrane electric field along with chloride and two protein-bound positive charges, resulting in an electroneutral translocation event. This idea will be tested in several ways, by using both chemically modified human band 3 and transgenic mice expressing band 3 mutated at this residue. The methods to be used include substrate binding measurements, pre-steady state and steady state tracer flux measurements, and patch-clamp electrical recordings. The second aim is to use improved chromatographic methods for analyzing hydrophobic peptides to finish full topological map of band 3, based on the positions of lysine residues. These studies should resolve current controversies regarding band 3 topology. The third aim is to use published methods to express the band 3 membrane domai in yeast (Saccharomyces cerevisiae), for the purpose of functional analysis of the site-directed mutations. The fourth aim is to identify, using both biochemical methods and mutagenesis, a second carboxyl group (other than E681) associated with the transport pathway and to localize exofacial lysine residue involved in subunit contacts. The mutagenesis will be performed in band 3 expressed in both Xenopus oocytes and in yeast.