ERYTHROCYTE MEMBRANE CYTOSKELETON ASSOCIATIONS

描述：(改编自调查人员摘要)在本提案中， 首席研究员打算做详细的分子生物学和 人红细胞锚定蛋白和人红细胞的生化研究 内收。红细胞锚蛋白的研究将包括结构分析 89 kDa结构域及其组成的33个残基的重复。已定义 89k Da结构域的区域将在细菌中表达，最小的 监视的本机二级结构所需的重复次数 圆二向色性将被确定。89k Da区域的晶体将 为X射线衍射法进行结构分析做好准备。装订 红细胞带3的33个残基重复区域内的位置 将映射属性域并将重复次数和唯一重复次数降至最低 确定绑定所需的。微管蛋白和钠钾结合部位的定位 将在89k Da结构域内进行ATPase结合。的域名 具有33个同源残基重复序列的其他蛋白质(果蝇Notch和C. 优雅的lin12基因产物)将被表达，并将用于 确定微管蛋白和其他可能的共同结合特异性 具有周期结构的大分子(核酸、磷脂)。 将绘制红细胞和脑粘连蛋白的血影蛋白结合部位图 以及能够表达血影蛋白结合的最小区域 活动已确定。这些粘连蛋白中的区域提供 红细胞血影蛋白的特异性，以及脑部血影蛋白的脑锚定蛋白， 将会被确定。假设Ankyrin的区域在 蛋白质2.2是选择性剪接的信使核糖核酸的产物，其功能是 将探索假底物和嵌段潜在的结合位点。 肾微粒体中已被证明识别的蛋白质 蛋白质2.2和未剪接的锚定蛋白将被鉴定。第二大调 这项研究的成分与人红细胞内收蛋白有关。完成 人APLHA和β亚基编码基因的克隆及序列分析 提出了红细胞内收蛋白的概念。这两个亚基都将被表达并 重组形成异二聚体和可能的同源二聚体。的活动站点 与钙调蛋白、肌动蛋白、血影蛋白/肌动蛋白、亚单位相互作用 杂二聚体和杂二聚体之间的缔合形成四聚体和 将使用映射技术来确定较高的低聚物。网站： 蛋白激酶A和C的磷酸化也将被测定。

DESCRIPTION: (Adapted from investigator's abstract) In this proposal, the principal investigator intends to do detailed molecular biology and biochemical studies of human erythrocyte ankyrin and human erythrocyte adducin. Studies on erythrocyte ankyrin will include structural analysis of the 89 kDa domain and its constituient 33-residue repeats. Defined regions of the 89 k Da domain will be expressed in bacteria and the minimal number of repeats required for native secondary structure as monitored by circular dichroism will be determined. Crystals of the 89 k Da domain will be prepared for analysis of structure by X-ray diffraction. The binding sites for erythrocyte band 3 within the 33 residue repeat region of this domain will be mapped and the minimal number of repeats and unique repeats required for binding determined. Mapping for sites of tubulin and Na/K ATPase binding within the 89 k Da domain will be performed. Domains of other proteins with homologous 33 residue repeats (Drosophila Notch and C. elegans lin12 gene products) will be expressed and they will be used to identify possible common binding specificity for tubulin and other macromolecules with periodic structures (nucleic acids, phospholipids). The spectrin binding sites of red cell and brain ankyrins will be mapped and the minimum regions competent for expressing spectrin-binding activities determined. The regions within these ankyrins that provide specificity for red cell spectrin, and brain ankyrin for brain spectrin, will be determined. The hypothesis that the region of ankyrin deleted in protein 2.2, a product of alternatively spliced mRNA, functions as a pseudosubstrate and blocks potential binding sites will be explored. Proteins in kidney microsomes that have been demonstrated to recognize protein 2.2 and not unspliced ankyrin will be identified. The second major component of the study relates to human erythrocyte adducin. Complete cloning and sequencing of cDNA encoding aplha and beta subunits of human erythrocyte adducin are proposed. Boths subunits will be expressed and recombined to form heterodimers and possibly homodimers. Active sites for interaction with calmodulin, actin, spectrin/actin, subunit interactions to heterodimers and associations between heterodimers to form tetramers and higher oligomers will be determined using mapping techniques. Sites of phosphorylation by protein kinases A and C will also be determined.