BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION TRANSPORT

红细胞阴离子转运的生物化学研究

• 批准号: 3274297
• 负责人: MICHAEL L JENNINGS
• 金额: $ 8.01万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 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 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274297
• 关键词: acid base balance; anions; bicarbonates; chemical structure function; chymotrypsin; erythrocyte membrane; human tissue; ion transport; membrane permeability; monoclonal antibody; papain; proteolysis; renal medulla; tissue /cell culture

The long-term goal of the proposed studies is to understand the molecular mechanisms and cellular functions of coupled Cl-HCO3 exchange across the plasma membranes of mammalian cells. This transport process is related to the larger problem f acid-base regulation, both on the cellular and whole-organism level. In the human red blood cell, Cl-HCO3 exchange is catalyzed by the major integrated membrane protein known as band 3. The topographical arrangement of the band 3 polypeptide in the membrane will be investigated by in situ proteolysis; the studies will be made feasible by the use of monoclonal antibodies directed against defined portions of the sequence. We recently developed a method for the labeling and conversion of functionally important extracellular glutamate side chains in band 3 to primary alcohols. A major goal of the proposed work is to localize these glutamate residues in the (known) band 3 sequence and to characterize the role of these residues in the anion translocation event, proton-anion cotransport, and the permeability barrier to conductive anion flux through the protein. The extracellular substrate anion binding site on band 3 is believed to be an arginine side chain. This side chain will be chemically modified with cyclohexanedione, and the labeled residue will be localized in the sequence by isolating the modified protein, digesting with protease to small peptides, and affinity- purifying the peptide of interest on immobilized borate derivative. Mammalian renal medullary collecting duct intercalated cells have a membrane protein that is immunologically related to red cell band 3. We will use monoclonal antibodies to isolate the homologous protein from bovine kidney. The protein will be reconstituted into lipid vesicles, and its possible function as a Cl- HCO3 exchanger will be investigated by a pH equilibration method. Other than for red cells and epithelia involved in acid or base secretion, it is not known whether Cl-HCO3 exchange has a general cellular function. Two human cell lines, K562 and HL60, are both known to have coupled Cl exchange; both lines also express the gene for a non-erythroid band 3 homolog, function of which is unknown. We will determine whether or not the Cl-Cl exchanger in these cells is likely to function physiologically as a Cl=HCO3 exchanger.

拟议研究的长期目标是了解 耦合 Cl-HCO3 的分子机制和细胞功能 跨哺乳动物细胞质膜的交换。 这 运输过程与酸碱这个更大的问题有关 细胞和整个生物体水平上的调节。 在人红细胞中，Cl-HCO3 交换由以下物质催化： 主要整合膜蛋白，称为带 3。 带 3 多肽的拓扑排列 将通过原位蛋白水解研究膜；研究 通过使用单克隆抗体定向将变得可行 针对序列的定义部分。 我们最近开发了一种标记和转换方法 功能重要的细胞外谷氨酸侧链 带3为伯醇。 拟议工作的一个主要目标是 将这些谷氨酸残基定位在（已知）带 3 中 序列并表征这些残基在 阴离子易位事件、质子-阴离子协同转运以及 传导性阴离子通量通过蛋白质的渗透性屏障。 带 3 上的细胞外底物阴离子结合位点是 据认为是精氨酸侧链。 该侧链将是 用环己二酮进行化学修饰，并标记 通过分离修饰的残基将定位在序列中 蛋白质，用蛋白酶消化成小肽，并进行亲和力- 在固定化硼酸盐上纯化感兴趣的肽 衍生物。 哺乳动物肾髓质集合管嵌入细胞 具有与红色免疫相关的膜蛋白 细胞带3.我们将使用单克隆抗体来分离 来自牛肾的同源蛋白。 蛋白质将是 重组为脂质囊泡，及其作为 Cl- 的可能功能 HCO3 交换剂将通过 pH 平衡进行研究 方法。 参与酸或碱​​的红细胞和上皮细胞除外 分泌，目前尚不清楚 Cl-HCO3 交换是否具有 一般细胞功能。 两种人类细胞系 K562 和 HL60， 已知两者都具有耦合的 Cl 交换；两条线也 表达非红细胞带 3 同源物的基因，功能 这是未知的。 我们将确定 Cl-Cl 是否 这些细胞中的交换器可能在生理上发挥着 Cl=HCO3 交换剂。