STR & EXP OF RED CELL MEMBRANE BAND 3

STR

基本信息

批准号：
3345545
负责人：
JOSEPH A WALDER
金额：
$ 12.36万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-12-01 至 1990-06-30
项目状态：
已结题

项目摘要

Band 3 is the major membrane spanning protein of the human erythrocyte, present at one million copies/cell, where it functions to mediate the exchange of chloride and bicarbonate across the lipid bilayer. The NH2-terminal 43,000 daltons of the protein forms a hydrophilic domain at the cytoplasmic surface of the red cell membrane which can be isolated by mild proteolytic digestion. This portion of the protein serves as an important attachment point for cytoskeleton to the erythrocyte membrane as well as a binding site for hemoglobin and several of the glycolytic enzymes within the red cell. The principal goals of the research proposed in this application are: 1) to determine the structure of the cytoplasmic domain of band 3; 2) to characterize the nature of the interaction of hemoglobin and the glycolytic enzymes with band 3; 3) to clone and sequence the cDNA for band 3; and 4) to use oligonucleotide probes, and later the cDNA, to prove a human genomic library for the gene for band 3 and hemologous proteins which may interact with the cytoskeleton in other tissues. Recently, we have directly shown that the acidic NH2-terminal sequence of band 3 is the binding site for hemoglobin. A synthetic peptide corresponding to the first 11 residues of band 3, AcM-E-E-L-Q-D-D-Y-E-D-E, as well as the intact cytoplasmic domain, were found to bind preferentially to deoxyhemoglobin. X-ray studies of the deoxyhemoglobin-peptide complex showed that the peptide binding site includes the binding site for 2,3-diphosphoglycerate (DPG) and, in addition, extends deeply 18 angstroms into the central cavity between the Beta subunits. Further experiments are proposed to determine the relative affinity of deoxyhemoglobin and several of the glycolytic enzymes for band 3 under physiological conditions, to determine the effect of pH, ionic strength, and competing metabolites (e.g. DPG) on the hemoglobin-band 3 interaction, to image the stereochemistry of the peptide-Beta chain contacts at high resolution, and finally to crystallize and determine the structure of the cytoplasmic domain of band 3. Our studies of the molecular biology of band 3 are aimed at pursuing the hypothesis, suggested by immunological studies, that membrane proteins in other tissues may interact with the cytoskeleton through segments of the polypeptide homologous with the cytoplasmic portion of band 3. These studies also will provide the basis for investigation of the regulated expression of band 3 during erythroid development.

带3是人类红细胞的主要膜蛋白质，以一百万份/单元的形式存在，它起作用以介导在整个脂质双层中交换氯化物和碳酸氢盐。这 NH2-末端43,000个蛋白质形成一个亲水性结构域红细胞膜的细胞质表面，可以通过轻度蛋白水解消化。蛋白质的这一部分用作细胞骨架上的重要附着点，以呈红细胞膜以及血红蛋白和几种糖酵解酶的结合位点在红细胞内。这项研究的主要目标应用是：1）确定细胞质结构域的结构乐队3; 2）表征血红蛋白相互作用的性质带有带3的糖酵解酶； 3）克隆并测序cDNA 对于频段3； 4）使用寡核苷酸探针，然后是cDNA 证明了带3个基因的人类基因组库和血液志的基因库可能与其他组织中细胞骨架相互作用的蛋白质。最近，我们直接表明带3是血红蛋白的结合位点。合成肽对应于频段3的前11个残基，ACM-E-E-E-l-Q-d-d-d-y-e-d-e，以及完整的细胞质结构域优先结合脱氧血红蛋白。脱氧血红蛋白肽复合物的X射线研究表明肽结合位点包括 2,3-二磷酸甘油（DPG），此外，还深深地延伸了18埃进入β亚基之间的中心空腔。进一步的实验是提议确定脱氧血红蛋白的相对亲和力和几个在生理条件下的带3的糖酵解酶确定pH，离子强度和竞争代谢产物的影响（例如 DPG）在血红蛋白波段3相互作用上，以形象形象高分辨率的肽β链链接触，最后结晶并确定带的细胞质结构域的结构 3。我们对频段3分子生物学的研究旨在追求免疫学研究表明的假设是膜蛋白在其他组织中，可以通过细分市场与细胞骨架相互作用带有带3的细胞质部分的多肽同源。研究还将为调查的调查提供基础红系发育过程中带3的表达。