STRUCTURE & FUNCTION OF HUMAN RBC MEMBRANE PROTEIN 7.2B

人红细胞的带7蛋白(相对分子质量26-30kda)构成 约占膜蛋白质量的3.4%。其中一种蛋白质(蛋白质 7.2B)在所有高加索人病例的红细胞中缺失 遗传性口腔炎，一种单价阳离子通透性障碍 这会导致慢性溶血性贫血。尽管这一发现 提示蛋白质7.2b可能是红细胞离子通道的一部分，其 实际功能目前尚不清楚。的目标和具体目标 这一建议是：1)了解蛋白质7.2b的结构和它的 红细胞膜内的形态结构，2)评估其推测的 用作离子通道的组件，以及3)探索计时 以及红细胞祖细胞中其生物合成和其生物合成机制 插入到膜脂双层中。为了实现这些目标， 将从红系组织中鉴定出蛋白质7.2b和c DNA 免疫或通过使用噬菌体表达的寡核苷酸 在E.Coli的图书馆。阳性克隆将被测序(Sanger)以 确定核苷酸序列(并由此确定氨基酸序列) 蛋白质7.2b的整个编码区。氨基酸序列将 通过化学方法和已知结构的位置进行验证 测定了修饰(磷酸化、棕榈化)。平行 遗传性口腔细胞的蛋白质和核酸研究有望实现 来解释为什么这些细胞中没有蛋白质7.2b。 蛋白质7.2b在单价阳离子转运中的可能作用是 通过1)7.2b的结构与已知结构的比较进行评估 运输蛋白，2)修饰蛋白7.2b的功能 多价抗7.2b抗体掺入完整红细胞的研究 使用渗透裂解技术和再密封与后续 对86Rb转运的评估，以及3)将蛋白7.2b导入 随后评价脂质体对阳离子转运的影响。蛋白 7.2B功能也将在非红系细胞中进行评估。时间，时间 蛋白7.2b在红系成熟过程中的表达及其意义 插入质膜将通过以下方式确定 免疫荧光显微镜和Northern印迹分析，寻找 7.2bmRNA的存在。这项工作的意义在于它 确定阳离子转运系统分子基础的可能性 和一种与溶血性贫血有关的疾病 天哪。

The band 7 proteins (MW 26-30 kda) of the human erythrocyte constitute about 3.4% of the membrane protein mass. One of the proteins (protein 7.2b) is missing from the erythrocytes of all Caucasian cases of hereditary stomatocytosis, a disorder of monovalent cation permeability that gives rise to chronic hemolytic anemia. Although this finding suggests that protein 7.2b may be part of an erythrocyte ion channel, its actual function is currently unknown. The objective and specific aims of this proposal are 1) to understand the structure of protein 7.2b and its topography within the erythrocyte membrane, 2) to evaluate its putative function as a component of an ion channel, and 3) to explore the timing and mechanics of its biosynthesis in erythrocyte progenitors and of its insertion into the membrane lipid bilayer. To achieve these goals, protein 7.2b c DNA from erythroid tissues will be identified immunologically or by oligonucleotides using a bacteriophage expression library in E. Coli. Positive clones will be sequenced (Sanger) to determine the nucleotide sequence (and from this the amino acid sequence) of the entire coding region of protein 7.2b. The amino acid sequence will be verified by chemical methods and the sites of known structural modification (phosphorylation, palmitylation) determined. Parallel protein and nucleic acid studies in hereditary stomatocytes are expected to yield an explanation for the absence of protein 7.2b in these cells. The possible role of protein 7.2b in monovalent cation transport will be evaluated by 1) comparison of the structure of 7.2b to that of known transport proteins, 2) modification of protein 7.2b function by incorporation of polyvalent anti-7.2b antibody into intact red cells using the technique of osmotic lysis and resealing with subsequent evaluation of 86Rb transport, and 3) introduction of protein 7.2b into liposomes followed by evaluation of effects on cation transport. Protein 7.2b function will also be evaluated in non-erythroid cells. The time of appearance of protein 7.2b during erythroid maturation as well as its insertion into the plasma membrane will be determined by immunofluorescent microscopy and by Northern blot analysis, searching for the presence of 7.2b mRNA. The significance of this work lies in its potential for defining the molecular basis of a cation transport system in the erythrocyte and of a disease associated with hemolytic anemia in man.