ANION EXCHANGERS IN PROXIMAL TUBULES

氯离子在肾脏近端小管的重吸收机制是 人们对此知之甚少。在红细胞中，运输的主要机制 穿过膜的氯化物通过阴离子交换带3 调解一种氯化物与一种重碳酸盐的交换。基于 转运活性和抑制剂敏感性，不同亚型 已在肾脏近端小管中发现了阴离子交换器。 其中包括氯化物/甲酸盐、氯化物/草酸盐、 氯/羟基，可能还有氯/碳酸氢盐交换剂。那里 关于阴离子交换剂的分子性质的信息很少 肾脏近端小管中的蛋白质(S)。阴离子交换传输 通常情况下，系统共享带3蛋白的几个特性 包括对二磺酸基二苯乙烯、丙磺舒、 呋塞米和精氨基修饰剂苯乙醛。 使用二磺酸二苯乙烯(DNS)亲和层析，我们有 阴离子交换法鉴定和部分纯化一种蛋白质 兔肾刷状缘膜的分离特性 近端小管。该蛋白分子量为162kD。这个 162 kD蛋白对已知的带3和带3的抑制物敏感 显示重组囊泡中的阴离子交换器活性。孵化 DNDS与蛋白激酶A和三磷酸腺苷的亲和纯化蛋白 导致重组脂质体的氯-氯交换活性降低。 利用阴离子对162kD蛋白进行了纯化，得到了均一的蛋白 交换(Pac Q)和尺寸排除层析。脂质体 用纯化的162 kDa蛋白重组后显示阴离子交换 ~(36)Cl-Cl交换法测定活性。该计划的主要目标 目前的建议是对功能、细胞和 162kD蛋白的分子性质。具体地说，162 KD 蛋白质将根据1.功能同一性进行评估。2. 信号转导。3.组织分布；4.分子 结构和编码的cDNA.纯化的162kD蛋白将是 重组成脂质体。162KD的功能同一性 然后，蛋白质将通过分析不同的 放射性标记的阴离子进入与162kD蛋白重组的脂质体中。 信号转导通路在近端调控中的作用 管状阴离子交换器的研究将通过孵育部分或 含有ATP和各种激酶的完全纯化的蛋白质随后 重建。然后对脂质体进行阴离子交换检测。 活动。针对162kD的多克隆抗体已经被提出 凝胶中162kD条带被切断的豚鼠的蛋白质。这个 将用抗体研究162kD的组织分布 蛋白质的蛋白质印迹分析和探索肾脏的c DNA文库 编码162kD蛋白的克隆。162kD的蛋白质被切断， 消化、提纯、气相测序机处理。二 获得了新的部分氨基酸序列。寡核苷酸 长度分别为23个和21个碱基的 多肽序列，并将用于探测肾脏的cdna文库 编码162kD蛋白的克隆。编码全长cDNA会 被克隆到表达载体中，并将用于转染COS 细胞。162KD的特性和功能特性 蛋白质将通过测量内流在转基因细胞中进行研究。 这些细胞中放射性标记的阴离子。建议的研究结果 研究应该显著增强我们对细胞的理解 和肾脏近端阴离子交换剂S的分子性质 小管。

The mechanisms of chloride reabsorption in the kidney proximal tubule are poorly understood. In red blood cells, the major mechanism for transport of chloride across the membrane is via the anion exchanger band 3 which mediates the exchange of one chloride for one bicarbonate. Based on the transport activity and inhibitors sensitivity, different isoforms of anion exchangers have been identified in the kidney proximal tubule. These, among others, include chloride/formate, chloride/oxalate, chloride/hydroxyl, and possibly chloride/bicarbonate exchangers. There is little information on the molecular properties of the anion exchanger protein(s) in the kidney proximal tubule. Anion exchange transport systems in general share several properties of the band 3 protein including sensitivity to inhibition by disulfonic stilbenes, probenecid, furosemide, and arginyl amino group modifier phenyglyoxal. Using disulfonic stilbene (DNS) affinity chromatography, we have identified and partially purified a protein with anion exchange characteristics in brush border membranes isolated from rabbit kidney proximal tubules. This protein has a molecular weight of 162 kD. The 162 kD protein is sensitive to the known inhibitors of the band 3 and displays anion exchanger activity in reconstituted vesicles.Incubation of the DNDS affinity-purified proteins with protein kinase A and ATP resulted in decreased Cl-Cl exchange activity in reconstituted liposomes. The 162 kD protein has been purified to homogeneity utilizing anion exchange (Pac Q) and size exclusion chromatography. The liposomes reconstituted with the purified 162 kDa protein displayed anion exchange activity as assayed by 36Cl-Cl exchange. The major objectives of the current proposal are characterization of functional, cellular, and molecular properties of the 162 kD protein. Specifically, the 162 kD protein will be evaluated with regard to 1. Functional identity. 2. Signal transduction. 3. Tissue distribution, and 4. Molecular structure and the encoding cDNA. The purified 162 kD protein will be reconstituted into liposomes. The functional identity of the 162 kD protein will then be determined by assaying the influx of different radiolabeled anions into liposomes reconstituted with the 162 kD protein. The signal transduction pathways involved in regulating the proximal tubule anion exchanger will be studied by incubating the partially or completely purified protein with ATP and various kinases followed by reconstitution. The liposomes will then be assayed for anion exchange activity. Polyclonal antibodies have been raised against the 162 kD protein in guinea pigs with the 162 kD band cut out of the gel. The antibody will be used to study the tissue distribution of the 162 kD protein by Western blot analysis and probe a kidney cDNA library for the clones encoding the 162 kD protein. The 162 kD protein was cut out, digested, purified, and subjected to gas phase sequencing machine. Two novel partial amino acid sequences have been obtained. Oligonucleotides of 23 and 21 bases in length have been synthesized according to the peptide sequences and will be used to probe a kidney cDNA library for the clones encoding the 162 kD protein. The encoding full length cDNA will be cloned into an expression vector and will be used to transfect COS cells. The characteristics and functional properties of the 162 kD protein will be studied in the transfected cells by measuring the influx of radiolabeled anions in these cells. The results of the proposed studies should significantly enhance our understanding about the cellular and molecular properties of the anion exchanger(s) in the kidney proximal tubule.