BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION TRANSPORT

拟议研究的长期目标是了解 Cl-HCO 3偶联的分子机制和细胞功能 通过哺乳动物细胞的质膜进行交换。 这 运输过程与更大的酸碱问题有关 调节，无论是在细胞和整个有机体水平。 在人红细胞中，Cl-HCO 3交换由以下物质催化： 主要的整合膜蛋白被称为带3。 的 带3多肽在细胞中的拓扑排列 膜将通过原位蛋白水解进行研究;研究 将通过使用单克隆抗体定向 与序列的定义部分进行比较。 我们最近开发了一种方法， 功能重要的细胞外谷氨酸侧链， 带3为伯醇。 拟议工作的主要目标是 将这些谷氨酸残基定位在（已知的）带3中 序列，并表征这些残基在 阴离子易位事件，质子-阴离子共转运，以及 渗透屏障，以传导阴离子通量通过蛋白质。 带3上的胞外底物阴离子结合位点是 被认为是精氨酸侧链。 这条侧链将是 用环己二酮化学修饰， 通过分离修饰的氨基酸残基， 蛋白质，用蛋白酶消化成小肽，和亲和- 在固定的硼酸盐上纯化感兴趣的肽 衍生物 哺乳动物肾髓质集合管闰细胞 有一种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 牛肾同源蛋白。 蛋白质将 重组成脂质囊泡，其可能的功能，作为一个氯- HCO 3交换器将通过pH平衡进行研究 法 除了红细胞和上皮细胞涉及酸或碱 分泌，尚不清楚Cl-HCO 3交换是否具有 一般细胞功能。 两种人类细胞系，K562和HL 60， 都已知具有耦合Cl交换;两条线也 表达非红细胞带3同源物的基因，功能为 这是未知的。 我们将决定氯氯是否 这些细胞中的交换器可能在生理上起着 Cl= HCO 3交换器。

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.