STRUCTURE/FUNCTION STUDY OF NA+/K+ ATPASE

NA /K ATP酶的结构/功能研究

• 批准号: 2378782
• 负责人: LOIS K LANE
• 金额: $ 28.04万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378782
• 关键词: 3T3 cells; HeLa cells; animal genetic material tag; catalyst; chemical binding; complementary DNA; enzyme mechanism; intracellular transport; ligands; molecular cloning; mutant; northern blottings; ouabain; phosphorylation; protein folding; protein sequence; protein structure function; rubidium; site directed mutagenesis; sodium potassium exchanging ATPase; synthetic nucleic acid; transfection; western blottings

The long-term goal of the proposed research is to elucidate the structure- function relationship of the mammalian Na.K-ATPase, with respect to both catalytic activity and cardiac glycoside binding. The specific aims of this proposal are to identify amino residues that are important for enzyme function in 1) the H7/h8 extracellular alpha subunit domain, which is postulated to be involved in both Na+/K+ and cardiac glycoside interaction; 2) the highly charged NH2-terminal alpha domain, which appears to be involved in K= deocclusion; and 3) to identify domains and residues that are involved directly and indirectly in the enzyme's interaction with ouabain and related drugs. The experimental approach for Aims 1 & 2 is to use site-directed mutagenesis and expression of the ouabain-insensitive rat alpha1 isozyme in HeLa cells, which contain an endogenous ouabain-sensitive isoform of Na.K- ATPase. Selection of stable clones with either ouabain or G418 enables us to quickly determine if any mutant enzyme is capable of catalyzing Na+/K+ transport or if it is "lethal". Active mutant enzymes will be functionally characterized with respect to Na.k-ATPase:E-P ratio. I50 for ouabain, 86Rb uptake, and K0.5's for ATP, Na+ and K+. The ability of inactive mutant enzymes (selected with G418) to be phosphorylated from ATP and Pi will also be measured. In addition, some mutant cDNA cassettes will be cloned into a modified rat alpha 1S cDNA, that encodes a ouabain-sensitive Na,K-ATPase (KD=1.57 nM), and expressed in ouabain-insensitive NIH/3T3 cells. This allows [3H] ouabain binding to be used as a tool for measuring the affinity of the mutant enzymes for ouabain, as well as the enzyme's affinity for those ligands that regulate ouabain binding (e.g., ATP. Pi, vanadate.Na+, K+ and Mg++). The comparison of functional parameters between wild type and mutant enzymes will define the roles that alpha subunit domains and specific residues play in th function of the Na.K-ATPase. These studies are currently ongoing in the applicant's laboratory. Good progress on specific aims 1 and 2 has already been achieved and initial studies on aim 3 are underway.

拟议研究的长期目标是阐明结构 - 哺乳动物Na.K-ATPase的功能关系 催化活性和心脏糖苷结合。 具体目的 该建议是确定对酶很重要的氨基残基 功能1）H7/H8细胞外α亚基域， 假定与Na+/K+和心脏糖苷相互作用有关； 2）高电荷的NH2末端alpha结构域，似乎是 参与k =去牙词； 3）确定域和残留物 直接和间接参与酶与 ouabain及相关药物。 目标1和2的实验方法是使用以网站为导向的 Ouabain不敏感的大鼠α1同工酶的诱变和表达 HeLa细胞，其中包含Na.K-的内源性ouabain敏感同工型 ATPase。 与ouabain或g418的稳定克隆一起选择使我们 快速确定任何突变酶是否能够催化Na+/K+ 运输或“致命”。 活性突变酶在功能上将是 以Na.K-ATPase：E-P比为特征。 i50 for ouaabain，86rb 吸收和K0.5用于ATP，Na+和K+。 非活性突变体的能力 从ATP和PI磷酸化的酶（选择G418）也将 可以测量。 此外，一些突变的cDNA盒将克隆到 修饰的大鼠alpha 1s cDNA，该cDNA编码ouabain敏感的Na，k-atpase （kd = 1.57 nm），并在对瓦巴因不敏感的NIH/3T3细胞中表达。 这 允许[3H] ouabain绑定用作测量亲和力的工具 瓦巴因的突变酶以及酶的亲和力 那些调节ouabain结合的配体（例如，atp。pi，vanadate.na+， K+和mg ++）。 野生型和突变体之间功能参数的比较 酶将定义α亚基域和特定的角色 残基在Na.K-ATPase的TH功能中发挥作用。 这些研究是 目前正在申请人的实验室中进行。 在特定方面取得了良好的进步 目的1和2已经实现，对目标3的初步研究是 进行。