BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION EXCHANGE

红细胞阴离子交换的生物化学研究

• 批准号: 6363218
• 负责人: MICHAEL L JENNINGS
• 金额: $ 20.5万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363218
• 关键词: Saccharomyces cerevisiae; Xenopus oocyte; band 3 protein; chloride ion; erythrocyte membrane; genetically modified animals; glutamates; high performance liquid chromatography; human subject; ion transport; laboratory mouse; lysine; protein sequence; protein structure function; site directed mutagenesis; sulfates; voltage /patch clamp

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.

描述：该项目的长期目标是了解 跨细胞膜耦合运输的分子机制。 焦点 该项目的重点是红细胞带 3 蛋白（也称为 AE1）， 它是红细胞膜的主要成分， 用于耦合传输机理研究的良好表征的模型系统。 除了作为模型系统的价值外，带 3 介导的氯-硫酸盐 交流（该项目的主要重点）具有潜在的相关性 DRA 介导的硫酸盐转运研究（在 腺瘤）蛋白质和负责发育不良的蛋白质， 软骨形成的遗传性疾病。 一般的做法是采取 利用丰富的带3来研究结构-功能关系 通过生化方法，并比较生化研究的结果 与通过定点诱变获得的那些。 第一个目标涉及 特定的谷氨酸残基（人 E681；小鼠 E699），其在 阴离子易位事件。 基于化学的工作假设 修饰实验，是这个残基上的负电荷 通常随着跨膜电场的大部分移动 氯离子和两个蛋白质结合的正电荷，导致 电中性易位事件。 这个想法将在多个方面进行测试 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带3。 所使用的方法包括 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性肽 根据赖氨酸的位置完成带 3 的完整拓扑图 残留物。 这些研究应该可以解决当前关于频带的争议 3 拓扑结构。 第三个目标是使用已发表的方法来表达频带3 酵母（酿酒酵母）中的膜域，目的是 定点突变的功能分析。 第四个目标是 使用生化方法和诱变鉴定第二个羧基 与转运途径相关的组（E681除外） 定位参与亚基接触的面外赖氨酸残基。 这 将在两个非洲爪蟾卵母细胞中表达的条带 3 中进行诱变 和酵母中。