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

描述(改编自申请人的摘要)：本报告的目的 建议分析结构与功能的关系 Na，K-ATPase。具体目的是：1.Na，K-ATPaseα/β亚基 互动。申请人将尝试确定哪些组合 α亚基和β亚基异构体可以通过在 编码α和β亚基亚基异构体并表达其结构的cDNA 在哺乳动物细胞中。2.α亚基的结构-功能。这个 申请者将绘制阿尔法亚单位的精细化地形图 通过创建无半胱氨酸的阿尔法亚基突变体来合成多肽。半胱氨酸 残基将被顺序引入预测的胞外或 无半胱氨酸突变体的细胞质环，以及其拓扑结构 用生物素标记的硫醇-或测定单个半胱氨酸标记 巯基反应性试剂。第二个目标将是识别序列 在形成钠结合位点的阿尔法亚单位内。3.基因 环尾小鼠的分析：α2亚单位的功能。申请人 将尝试确定Loopail(LP)小鼠是否在 Na，K-ATPaseα2亚单位(Atp1a2)基因的序列或表达。 4.β3亚基的分子分析。申请人建议： 进一步鉴定最近发现的Na，K-ATPaseβ3亚基。这个 目的是分离和鉴定人类基因组的cDNADNA序列 小鼠β3亚基并研制用于细胞定位的抗体探针 学习。

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.