BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION EXCHANGE

描述：该项目的长期目标是了解 跨细胞膜耦合转运的分子机制。焦点 该项目的重点是红血球带3蛋白(也称为AE1)， 它是红细胞膜的主要成分，是一种 耦合输运力学研究的良好模型体系。 除了作为模型体系的价值外，带3介导的氯-硫酸盐 交换(本项目的主要重点)可能与以下内容相关 DRA介导的硫酸盐转运研究(下调在 腺瘤)蛋白和与营养不良有关的蛋白，以及 遗传性软骨形成障碍。一般的做法是采取 谱带3丰度在结构-功能关系研究中的优势 通过生化方法，比较生化研究的结果 与通过定点突变获得的基因进行比较。第一个目标涉及一个 特定的谷氨酸残基(人E681；小鼠E699)，它在 阴离子移位事件。基于化学物质的工作假说 修饰实验，就是把负电荷放在这个残留物上 通常会穿过大部分跨膜电场，并伴随着 氯化物和两个蛋白质结合的正电荷，导致 电子中和易位事件。这一想法将在几个方面进行测试 方法：同时使用化学修饰的人Band 3和转基因小鼠 表达带3在该残基发生突变。将使用的方法包括 衬底结合测量、稳态前和稳态示踪剂 磁通测量和膜片钳电子记录。第二个目标是 用改进的层析方法分析疏水性多肽 根据赖氨酸的位置完成带3的完整拓扑图 残留物。这些研究应该会解决目前关于BAND的争议 3拓扑学。第三个目标是使用已公布的方法来表示带3 酵母(酿酒酵母)的膜结构域，用于 定点突变的功能分析。第四个目标是 使用生化方法和诱变方法鉴定第二个羧基 与运输途径相关的组(E681除外)和TO 定位亚基接触中涉及的表面外赖氨酸残基。这个 在两个非洲爪哇卵母细胞中表达的第3条带将进行突变 还有酵母菌。

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.