RENAL OXALATE TRANSPORTER

肾结石每年困扰着大约50万美国人，其中75% 在所有肾结石中，草酸钙是主要成分。尽管它的 在肾结石发病机制中的重要作用，目前知之甚少 关于负责草酸的蛋白质的特性 沿肾单位排泄。我们最近已经溶解了 重组兔肾皮质的两个草酸转运体； 碱侧硫酸盐/草酸盐交换器和刷状边界膜(BBM) 氯化物/草酸盐交换剂。这项提议的目标是净化 重组BBM氯化物/草酸盐交换剂，并对其进行表征 蛋白质的结构和功能。 为了将重组草酸转运蛋白纯化到均一状态 拟议的净化方案将在即将完成的基础上进行扩展 已经实现的提纯。我们计划遵循 草酸转运体在每一步的功能活性 进行纯化，以确认目的蛋白已经 纯化并确保纯化的蛋白质保持在其原始状态 状态，以便描述其功能。建议采取的其他步骤 实现纯化的草酸转运蛋白包括凝集素、大小 排除层析和染料配基亲和层析。一旦 蛋白质已纯化成均一、多克隆和单克隆 就会产生抗体。这些细胞将被用于免疫亲和力。 层析，以简化纯化方案并 提高纯化蛋白的得率。抗体还将使我们能够 识别其他组织中的免疫相关蛋白以及 肾单位的不同节段。 草酸转运蛋白的纯化为我们提供了机会 在没有其他BBM蛋白的系统中检测其功能。这是 在草酸盐运输的情况下尤其重要，因为 具有重叠底物专一性的多个阴离子交换途径 近端小管的顶膜。功能表征 将包括鉴定其底物专一性、生电性和pH 依赖。草酸转运体与氯离子的关系 运输将被详细检查，因为最近的证据表明 草酸是氯化钠重吸收的重要中间体。 肾单位的不同节段。 将获得纯化蛋白的部分氨基酸序列， 并与抗体一起用于克隆cDNAs 编码肾草酸转运蛋白。完整的序列将 与通过酶消化获得的信息相结合 蛋白质及其糖基化的作用以预测其次级 结构。通过纯化草酸转运蛋白，鉴定其 功能，并将其克隆到细胞系统中进行表达 在未来有机会开始将 蛋白质及其功能。希望这些类型的研究 将有助于理解草酸转运在 肾结石和盐分在肾脏的重吸收。

Kidney stones afflict approximately 500,000 Americans each year, with 75% of all renal stones composed primarily of calcium oxalate. Despite its importance in the pathogenesis of nephrolithiasis, very little is known about the characteristics of the proteins responsible for oxalate excretion along the nephron. We have recently solubilized and reconstituted two oxalate transporters from the rabbit renal cortex; the basolateral sulfate/oxalate exchanger and the brush border membrane (BBM) chloride/oxalate exchanger. The objective of this proposal is to purify the reconstituted BBM chloride/oxalate exchanger and to characterize the protein in terms of its structure and function. In order to purify the reconstituted oxalate transporter to homogeneity the proposed purification protocol will expand upon the nearly complete purification that has already been achieved. We plan on following the functional activity of the oxalate transporter during each step of purification in order to confirm that the protein of interest has been purified and to assure that the purified protein remains in its native state in order to characterize its function. Additional steps proposed to achieve purification of the oxalate transporter include lectins, size exclusion chromatography and dye-ligand affinity chromatography. Once the protein has been purified to homogeneity, polyclonal and monoclonal antibodies will be generated. These will in turn be used in immunoaffinity chromatography in order to simplify the purification protocol and to increase the yield of purified protein. Antibodies will also enable us to identify immunologically related proteins in other tissues as well as in various segments of the nephron. Purification of the oxalate transporter provides us with the opportunity to examine its function in a system free of other BBM proteins. This is particularly important in the case of oxalate transport because of the multiple anion exchange pathways with overlapping substrate specificity on the apical membrane of the proximal tubule. Functional characterization will include identifying its substrate specificity, electrogenicity and pH dependence. The relationship of the oxalate transporter to chloride transport will be examined in detail because of recent evidence suggesting that oxalate is an important intermediate in the reabsorption of NaCl in different segments of the nephron. A partial amino acid sequence will be obtained for the purified protein, and in conjunction with the antibodies, will be used to clone the cDNA encoding the renal oxalate transport protein. The complete sequence will be combined with information obtained from enzymatic digestion of the protein and the role of glycosylation in order to predict its secondary structure. By purifying the oxalate transporter, characterizing its function and cloning the cDNA with expression into a cell system we will have the opportunity in the future to begin correlating the structure of the protein with its function. It is hoped that these types of studies will lead to an understanding of the role of oxalate transport in nephrolithiasis and in salt reabsorption in the kidney.