BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION TRANSPORT

拟议研究的长期目标是了解 偶联氯-HCO3的分子机理和细胞功能 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 无论是在细胞层面还是在整个生物体层面上的调控。 在人类红细胞中，氯-HCO3交换是由 主要的整合膜蛋白称为带3。 条带3多肽在中国大陆的地形排列 膜将通过原位蛋白分解进行研究；研究 将通过使用定向的克隆抗体使其成为可能 与序列的定义部分进行比对。 我们最近开发了一种标记和转换的方法 具有重要功能的胞外谷氨酸侧链 伯醇的3带。拟议工作的一个主要目标是 将这些谷氨酸残基定位在(已知的)带3 序列，并表征这些残基在序列中的作用 阴离子转运事件、质子-阴离子共转运和 通过蛋白质的传导阴离子通量的渗透性屏障。 带3上的胞外底物阴离子结合部位为 据信是精氨酸侧链。这条侧链将是 用环己二酮进行化学修饰，并标记 残基将通过分离修饰的 蛋白质，用酶消化成小肽，以及亲和力- 固定化硼酸盐法提纯目的多肽 衍生品。 哺乳动物肾髓质集合管嵌入细胞 有一种与红色免疫相关的膜蛋白 细胞带3。我们将使用单抗来分离 牛肾中的同源蛋白。蛋白质将会是 重组成脂泡，及其可能的氯-功能 HCO3交换器将通过pH平衡进行研究 方法。 除了与酸或碱有关的红细胞和上皮细胞外 分泌，目前尚不清楚氯-HCO3交换是否具有 一般的细胞功能。两个人类细胞系，K562和HL60， 都已知具有耦合的氯交换；两条线还 表达非红系第3带同源物的基因，功能 这是个未知数。我们将确定氯-氯是否 这些细胞中的交换器很可能在生理上起到 CL=HCO3交换剂。

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.