MOLECULAR ANALYSIS OF NA+/K+-ATPASE

本提案的目的是应用以下技术： 分子遗传学对Na，K-ATP酶结构的分析， 功能和生物起源。我们已经开发了一种表达系统， 对克隆的啮齿动物Na，K-ATPase基因进行生物学活性检测。 该系统形成了旨在分析 ATP酶和控制的结构功能关系 ATP酶生物合成和组装的潜在机制。的 该提案的具体目标包括：1）Na，K- ATP酶与心脏糖苷。我们会评估 酶的结构和强心苷的结合之间的联系。 最初的实验将涉及嵌合cDNA的构建 分子之间的cDNA分子编码哇巴因抗性和 哇巴因敏感形式的Na，K-ATP酶，以描绘 与药物相互作用的α链区域。 我们将 然后使用定点诱变来改变 编码哇巴因敏感性ATP酶的cDNA， 编码的酶对哇巴因的抗性 2)亚细胞 Na，K-ATP酶同工酶的定位。 原位技术 杂交将被用来了解是否替代形式的 Na，K-ATP酶mRNA在相同或不同的细胞内表达 一种特殊的组织。 肽衍生的抗体特异于 每种ATP酶同工型将被开发并用于区分 细胞中的交替ATP酶同工型合成一种以上 同种型和鉴定亚细胞定位位点。目标 这些实验的目的是获得关于 异构体表达与功能的关系。第三章 影响Na，K-ATP酶生物合成的控制机制。 我们将 试图分离和表征可能是 负责组织特异性和发育调节 Na，K-ATP酶mRNA的表达。 最初的兴趣将集中在 启动子和其他5'调控区的鉴定 参与Na，K-ATP酶mRNA表达的调节。 我们将 我也试图鉴定α和β亚基内的区域 可能在转录后调控中发挥作用的mRNA 亚基生物合成的过程。4)β亚基与 Na，K-ATP酶功能 我们将尝试开发一种分析系统， 它通过产生一种 需要β亚基过表达的系统 用于细胞活力。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.