MOLECULAR ANALYSIS OF NA+/K+-ATPASE

该提案的目标是应用以下技术 分子遗传学对Na，K-ATPase结构的分析， 功能和生物发生。我们已经开发了一种表达系统来 检测克隆的啮齿动物Na，K-ATPase基因的生物学活性。 该系统构成了设计用来分析 ATPase与调控的结构功能关系 ATPase生物合成和组装的潜在机制。这个 该提案的具体目标包括：1)Na，K-的相互作用 含强心苷的ATPase。我们将评估这一关系 酶结构与心脏糖苷结合之间的关系。 初步实验将涉及构建嵌合的cdna。 编码哇巴因抗性的cdna分子和 哇巴因敏感形式的Na，K-ATPase 与药物相互作用的阿尔法链区域。我们会 然后使用定点突变来改变特定的残基 编码哇巴因敏感的ATPase的cDNA，旨在将 哇巴因抗性的编码酶。2)亚细胞 Na，K-ATPase亚型的定位。浅谈现场施工技术 杂交将被用来了解替代形式的 Na，K-ATPase mRNA在相同或不同的细胞中表达 一种特定的组织。特异性多肽衍生抗体 每一种ATPase亚型都将被开发并用于区分 合成多种ATPase异构体的细胞 并确定亚细胞定位的位置。目标是 这些实验中的一项是获得关于 异构体表达与功能的关系。3) 影响Na，K-ATPase生物合成的调控机制。我们会 试图分离和鉴定DNA序列，这可能是 负责组织特异性和发育调节 Na，K-ATPase mRNAs表达。最初的兴趣将集中在 启动子及其他5‘端控制区的鉴定 参与调节Na，K-ATPase基因的表达。我们会 还试图确定α和β亚基内的区域 可能在转录后调控中发挥作用的mRNAs 亚基生物合成。4)β亚基与 Na，K-ATPase功能。我们将尝试开发一种检测系统 它通过创建来测量8亚基的生物活性 一种需要过表达β亚基的系统 为了细胞的存活。然后，该系统将用于评估 β亚基的结构功能关系。

The objective of this proposal is to apply the techniques of molecular genetics to the analysis of Na,K-ATPase structure, function and biogenesis. We have developed an expression system to test the biological activity of cloned rodent Na,K-ATPase genes. This system forms the framework for experiments designed to analyze structure function relationships for the ATPase and control mechanisms underlying ATPase biosynthesis and assembly. The specific aims of the proposal include: 1) Interaction of the Na,K- ATPase with Cardiac Glycosides. We will evaluate the relationship between enzyme structure and the binding of cardiac glycosides. Initial experiments will involve the construction of chimeric cDNA molecules between cDNA molecules encoding ouabain resistant and ouabain sensitive forms of the Na,K-ATPase in order to delineate regions of the alpha chain which interact with the drug. We will then use site directed mutagenesis to alter specific residues in a cDNA encoding a ouabain sensitive ATPase in an effort to convert the encoded enzyme to ouabain resistance. 2) Subcellular Localization of Na,K-ATPase Isoforms. The technique of in situ hybridization will be used to learn whether alternative forms of Na,K-ATPase mRNA are expressed within the same or different cells of a particular tissue. Peptide derived antibodies specific for each ATPase isoform will be developed and used to distinguish alternative ATPase isoforms in cells synthesizing more than one isoform and to identify sites of subcellular localization. The goal of these experiments will be to obtain basic information regarding the relationship between isoform expression and function. 3) Control Mechanisms Affecting Na,K-ATPase Biosynthesis. We will attempt to isolate and characterize DNA sequences which may be responsible for the tissue specific and developmentally regulated expression of Na,K-ATPase mRNAs. Initial interest will focus on identification of the promoter and other 5' control regions involved in the regulation of Na,K-ATPase mRNA expression. We will also attempt to identify regions within alpha and beta subunit mRNAs which may play a role in the postranscriptional regulation of subunit biosynthesis. 4) Relationship of the beta Subunit to Na,K-ATPase Function. We will attempt to develop an assay system which measures the biological activity of the 8 subunit by creating a system in which overexpression of the beta subunit is required for cell viability. This system will then be used to assess structure function relationships for the beta subunit.