CARDIAC NA,K-ATPASE GENES: STRUCTURE AND FUNCTION

这项提议的目标是应用分子生物学技术 从遗传学到结构功能关系的分析 Na，K-ATPase。我们已经开发了一个表达系统来测试 克隆的Na，K-ATPase基因的生物学活性。这一系统构成了 设计实验框架分析两者之间的关系 酶的结构和功能。具体目标包括：1)Na，K-ATPase α/β亚基相互作用。我们将尝试确定哪一个 α和β亚基的组合可以组装成全酶。 为了解决这个问题，我们将使用表位添加来标记一个cdna。 编码特定的α和β亚基亚基异构体。介绍了 构建成CV-1细胞并免疫沉淀表达融合蛋白 带有针对表位标签的抗体的蛋白质将允许我们 确定产生哪些α/β亚基。这种方法应该 也使我们能够研究转基因细胞中的同工酶功能。2) 结构--阿尔法亚单位的功能。我们会尝试找出 引起Na+亲和力差异的序列 α1和α3亚基亚基异构体。的构造和表达 Alpha1和Alpha3亚单位之间的嵌合体应该允许 确定α亚基内与Na+相互作用的位置 并有助于Na+结合。第二种方法将被设计为 分析α2亚型的生化性质。这些 实验应该能让我们更清楚地了解 三种α亚基亚基异构体之间的功能关系。3) β亚基的功能。为了分析β亚基的作用， 我们将利用这样一个事实，即编码β亚基的cdna H，K-ATPase可通过以下途径赋予灵长类细胞哇巴因抗性 转染法。我们将使用表位加法来确定H，K- ATPaseβ亚基可与Na，K-ATPaseα亚基组装。这个 嵌合H，K-/Na，K-ATPaseβ亚基的构建及表达 CDNA应该允许我们识别β亚基中的 助长了哇巴因抵抗运动。β亚基的表达 提供了一个学习结构函数关系的机会 表达初级氨基酸序列改变的β亚基。 这些实验也具有实际意义，因为使用了 强心苷在治疗充血性心力衰竭中的作用。

The objective of this proposal is to apply the techniques of molecular genetics to the analysis of structure function relationships for the Na,K-ATPase. We have developed an expression system to test the biological activity of cloned Na,K-ATPase genes. This system forms the framework for experiments designed to analyze the relationship between enzyme structure and function. Specific aims include: 1) Na,K-ATPase alpha/beta subunit interaction. We will attempt to determine which combinations of alpha and beta subunits can assemble to form holoenzyme. To address this issue, we will use epitope addition to tag a cDNA encoding a specific alpha and beta subunit isoform. Introduction of the construct into CV-1 cells and immunoprecipitation of the expressed fusion protein with an antibody against the epitope tag will allow us to determine which alpha/beta subunits are produced. This approach should also allow us to study isoenzyme function in transfected cells. 2) Structure-Function of the alpha subunit. We will attempt to identify sequences responsible for the variation in Na+ affinity between the alpha1 and alpha3 subunit isoforms. Construction and expression of chimeras between alpha1 and alpha3 subunit cDNAs should permit identification of sites within the alpha subunit that interact with Na+ and contribute to Na+ binding. A second approach will be designed to analyze the biochemical properties of the alpha2 isoform. These experiments should allow us to derive a clearer understanding of the functional relationships among the three alpha subunit isoforms. 3) Function of the beta subunit. To analyze the role of the beta subunit, we will take advantage of the fact that a cDNA encoding the beta subunit of the H,K-ATPase can confer ouabain resistance to primate cells by transfection. We will use epitope addition to determine if the H,K- ATPase beta subunit can assemble with the Na,K-ATPase alpha subunit. The construction and expression of chimeric H,K-/Na,K-ATPase beta subunit cDNAs should permit us to identify sites within the beta subunit that contribute to ouabain resistance. Expression of the beta subunit provides an opportunity to study structure function relationships by expressing beta subunits with alterations in primary amino acid sequence. These experiments also have practical significance because of the use of cardiac glycosides in the treatment of congestive heart failure.