CARDIAC NA+/K+ ATPASE GENES--STRUCTURE/FUNCTION

描述（改编自申请人的摘要）： 建议分析结构与功能的关系 Na，K-ATP酶的活性。 具体目标是：1. Na，K-ATP酶α/β亚基 互动 申请人将尝试确定哪些组合 α和β亚单位同种型可以通过将表位标签插入到 编码α和β亚基同种型并表达构建体的cDNA 在哺乳动物细胞中。 2. α亚基的结构-功能。 的 申请人将开发α亚单位的精细地形图 通过产生无半胱氨酸的α亚基突变体来制备多肽。 半胱氨酸 残基将被顺序引入预测的细胞外或 细胞质环的半胱氨酸少突变体，和拓扑结构的 使用生物素标记的巯基或 巯基反应性试剂。 第二个目标将是确定序列 在α亚基内形成钠结合位点。 3. 遗传 环尾鼠的分析：α 2亚基的功能。 申请人 将试图确定环尾（Lp）小鼠是否表现出 Na，K-ATP酶α 2亚基（Atp 1a 2）基因的序列或表达。 4. β 3亚基的分子分析。 申请人建议 进一步表征最近发现的Na，K-ATP酶β 3亚基。 的 目的是分离和表征的cDNA和基因组DNA序列的 鼠β 3亚单位，并开发用于细胞定位的抗体探针 问题研究

DESCRIPTION (adapted from the applicant's abstract): The objective of this proposal is to analyze the relationship between the structure and function of Na,K-ATPase. The specific aims are: 1. Na,K-ATPase alpha/beta subunit interaction. The applicant will attempt to determine which combinations of alpha and beta subunit isoforms can associate by inserting epitope tags into cDNAs encoding alpha and beta subunit isoforms and expressing the constructs in mammalian cells. 2. Structure-function of the alpha subunit. The applicant will develop a refined topographical map of the alpha subunit polypeptide by creating a cysteine-less alpha subunit mutant. Cysteine residues will be sequential introduced into predicted extracellular or cytoplasmic loops of the cysteine-less mutant, and the topology of individual cysteine tags determined using biotin-labeled thiol- or sulfhydryl-reactive reagents. A second goal will be to identify sequences within the alpha subunit that form the sodium binding site. 3. Genetic analysis of Looptail mice:Function of the alpha2 subunit. The applicant will attempt to determine whether Looptail (Lp) mice exhibit alterations in the sequence or expression of the Na,K-ATPase alpha2 subunit (Atp1a2) gene. 4. Molecular analysis of the beta3 subunit. The applicant proposes to further characterize the recently discovered Na,K-ATPase beta3 subunit. The goal is to isolate and characterize cDNA and genomic DNA sequences for the murine beta3 subunit and develop antibody probes for cell localization studies.