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MOLECULAR BIOLOGY OF THE RENAL NA/H EXCHANGER

肾脏 NA/H 交换器的分子生物学

基本信息

批准号：
3086406
负责人：
ROBERT F REILLY
金额：
$ 9.35万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 1993-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3086406
关键词：
apical membrane genetic library genetic manipulation hydrogen ion transport laboratory mouse laboratory rabbit membrane transport proteins molecular cloning monoclonal antibody nucleic acid probes protein sequence recombinant DNA renal tubular transport renal tubule sodium structural genes

项目摘要

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.

该项目的总体目标是确定蛋白质，介导Na+-H+交换穿过管腔膜，近曲小管细胞，并克隆和测序的结构这个运输系统的基因。申办者的既往研究兔肾微绒毛共价标记实验室具有二环己基碳二亚胺的膜，甲基异丁基氨氯吡咪已经鉴定出至少两种不同的作为Na+-H+交换剂的候选物的多肽或其一种亚单位。共同发起人的实验室最近分离出了一种 LLC-PK 1肾细胞系，其缺乏或过表达Na+-H+交换活性。在拟议的研究中，共价标记的顶端膜囊泡分离自这些将进行突变细胞系，以确定哪一种先前鉴定的候选多肽实际上代表了 Na+-H+交换剂或其亚基之一。单克隆然后产生针对该多肽的抗体。特异性将通过使用复溶来确认抗体 Na+-H+交换活性测定，可参见申办方的实验室然后将这些抗体用于亲和纯化转运蛋白一个λ gt 11 cDNA表达文库先前在申办方实验室从家兔肾脏制备然后用抗转运蛋白的抗体筛选皮质蛋白质或基于以下构建的寡核苷酸探针对来自纯化蛋白质的肽进行部分测序。将对分离的克隆cDNA进行测序，蛋白质的结构，从而推断。后续研究将解决二级结构，作用机制，生物合成和Na+-H+交换的调节。因为质膜Na+-H+交换器是一种普遍存在的转运体系统涉及细胞生理学的各个方面，建议研究将具有普遍的生物医学意义。最重要的是，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、代单克隆抗体、蛋白质生物化学和重组 DNA技术