CHARACTERIZATION OF A RENAL OXALATE TRANSPORTER

肾结石每年折磨大约50万美国人，其中75%的人患有肾结石。 主要由草酸钙组成的肾结石。尽管 在肾结石发病机制中的重要性，知之甚少 负责草酸盐的蛋白质的特性 沿着肾单位沿着排泄。我们最近溶解了， 重建两个草酸转运体从兔肾皮质; 基底外侧硫酸盐/草酸盐交换体和刷状缘膜（BBM） 氯化物/草酸盐交换剂。这项提案的目的是净化 重组BBM氯化物/草酸盐交换剂，并表征 蛋白质的结构和功能。 为了将重组的草酸转运蛋白纯化至均一 所提出的纯化方案将在几乎完全的 已经实现的净化。我们计划按照 草酸转运蛋白的功能活性，在每一个步骤 纯化，以确认目的蛋白已经被纯化。 以确保纯化的蛋白质保持其天然状态， 国家，以体现其功能。拟议采取的其他步骤， 实现草酸转运蛋白的纯化，包括凝集素、大小 排阻色谱和染料-配体亲和色谱。一旦 蛋白质已被纯化到同质，多克隆和单克隆 将产生抗体。这些将反过来用于免疫亲和 通过色谱法纯化，以简化纯化方案， 提高了纯化蛋白的产量。抗体还将使我们能够 鉴定其他组织中的免疫相关蛋白， 肾单位的各个部分。 草酸转运蛋白的纯化为我们提供了 在没有其他BBM蛋白的系统中检测其功能。这是 在草酸盐转运的情况下特别重要，因为 具有重叠底物特异性的多种阴离子交换途径， 近端小管的顶膜。功能表征 将包括鉴定其底物特异性、产电性和pH 依赖草酸转运体与氯离子的关系 运输将被详细审查，因为最近的证据表明， 草酸盐是重吸收NaCl的重要中间体， 肾单位的不同部分。 将获得纯化蛋白的部分氨基酸序列， 并与抗体结合，将用于克隆cDNA 编码肾草酸转运蛋白。 完整的序列将 结合从酶消化的信息， 蛋白质和糖基化的作用，以预测其二级 结构通过纯化草酸转运蛋白，表征其 功能和克隆表达的cDNA到细胞系统中，我们将 在未来有机会开始将 蛋白质及其功能。希望这些研究 将有助于理解草酸盐转运在 肾结石和盐在肾脏中的重吸收。

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.