MOLECULAR BIOLOGY OF THE RENAL NA/H EXCHANGER

该项目的总体目标是确定蛋白质(S)， 介导(S)膜上Na~+-H~+交换 对近曲小管细胞进行克隆和测序。 这个运输系统的基因。赞助商之前的研究 兔肾微绒毛共价标记的实验室研究 含有二环己基碳二亚胺和 甲基异丁基阿米洛利已经鉴定出至少两种不同的 多肽作为Na+-H+交换器的候选者或其 亚单位。联合赞助商的实验室最近分离出了一种 LLC-PK1肾细胞系 过表达Na+-H+交换活性。在拟议的研究中， 分离的顶膜囊泡的共价标记 将进行突变细胞系的实验，以确定哪些 先前确定的候选多肽实际上代表 Na+-H+交换器或其一个亚基。单克隆 然后就会产生针对这种多肽的抗体。特异性 的抗体将通过重组的方式得到确认 赞助商提供的Na+-H+交换活性的测定 实验室。这些抗体将被用于亲和纯化 运输蛋白。一个lambda gtll基因表达文库 之前在赞助商的实验室从兔肾中制备 然后用抗体对大脑皮质进行筛选，以对抗运输 蛋白质或与构建在以下基础上的寡核苷酸探针 对来自纯化蛋白的多肽进行部分测序。 分离的克隆的cDNAs将被测序，并初步 由此推断出蛋白质的结构。后续研究将 解决二级结构、作用机制、 Na+-H+交换器的生物合成和调节。因为 质膜Na~+-H~+交换是一种无处不在的转运 系统涉及细胞生理学的多个方面，建议 研究将具有普遍的生物医学意义。多数 重要的是，该项目将提供各种 膜生理学和分子生物学相关技术 生物学包括组织培养、膜分离、传代 单抗、蛋白质生物化学和重组 DNA技术。

The overall aim of the project is to identify the protein(s) that mediate(s) Na+-H+ exchange across the luminal membrane of the proximal tubule cell, and to clone and sequence the structural gene for this transport system. Previous studies in the sponsor's laboratory employing covalent labeling of rabbit renal microvillus membranes with dicyclohexylcarbodiimide and methylisobutylamiloride have identified at least two different polypeptides as candidates for the Na+-H+ exchanger or one of its subunits. The co-sponsor's laboratory recently isolated mutant lines of LLC-PK1 kidney cells that are either deficient in or overexpress Na+-H+ exchange activity. In the proposed studies, covalent labeling of apical membrane vesicles isolated from these mutant cell lines will be performed to define which of the previously identified candidate polypeptides actually represents the Na+-H+ exchanger or one of its subunits. Monoclonal antibodies to this polypeptide will then be generated. Specificity of the antibodies will be confirmed by use of a reconstitution assay for Na+-H+ exchange activity available in the sponsor's laboratory. These antibodies will then be used to affinity purify the transport protein. A lambda gtll cDNA expression library previously prepared in the sponsor's laboratory from rabbit renal cortex will then be screened with antibodies against the transport protein or with oligonucleotide probes constructed on the basis of partial sequencing of peptides derived from the purified protein. Isolated cloned cDNA's will be sequenced and the primary structure of the protein thereby deduced. Subsequent studies will address the secondary structure, mechanism of action, biosynthesis, and regulation of the Na+-H+ exchanger. Because the plasma membrane Na+-H+ exchanger is a ubiquitous transport system involved in diverse aspects of cell physiology, the proposed studies will be of general biomedical significance. Most importantly, the project will provide training in a variety of techniques relevant to membrane physiology and molecular biology including tissue culture, membrane isolation, generation of monoclonal antibodies, protein biochemistry, and recombinant DNA technology.