NA PUMP--CHARACTERIZATION OF DIGITALIS BINDING SITE

NA 泵——洋地黄结合位点的表征

• 批准号: 2029104
• 负责人: KAI Y XU
• 金额: $ 11.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029104
• 关键词: digitalis; drug receptors; enzyme substrate complex; isozymes; laboratory rabbit; laboratory rat; ouabain; protein purification; protein structure function; receptor binding; receptor sensitivity; site directed mutagenesis; sodium potassium exchanging ATPase

DESCRIPTION (Adapted from the abstract): Na+, K+-ATPase (Na pump) is the target receptor for the biological actions of the digitalis. The precise mechanism and the detailed knowledge of which amino acid side-chain groups are responsible for digitalis binding have remained open questions. The focus of this proposal is to identify the region and the specific amino acids that participate in digitalis binding to the Na+, K+-ATPase and are essential for ion transport. The strategy of the study is to use native digitalis and its derivative with the complementary technology involving protein chemistry and molecular biology to provide direct evidence of a specific, covalently bound intermediate (enzyme-digitalis complex), that is undetectable by ordinary chemical analyses. Purified cardiac Na+, K+-ATPase from rabbit and rat will be used for the affinity labeling. The newly identified residues will guide the design of site-directed mutagenesis studies to further characterize and explore the digitalis binding sites in the three isoforms of the rat Na+, K+-ATPase. Five specific aims are proposed to achieve the objective: Aim 1, to identify the structural determinants at the digitalis binding pocket of both ouabain-sensitive (rabbit) and ouabain-resistant (rat) Na+, K+-ATPase, and to test the hypothesis that the newly identified drug binding sites are the universal digitalis binding sites at the protein level; Aim 2, to characterize the newly identified amino acids that reside in the digitalis binding sites at the molecular level, and to further test the hypothesis that these sites are the universal drug binding sites for three isoforms of the Na+, K+-ATPase; Aim 3, to determine whether the newly identified amino acids contribute to the ouabain-sensitivity,and to test whether the new drug binding sites are structurally near the substrate binding site in the extracellular domain of the Na+, K+, ATPase; Aim 4, to explore the functional relationship between the neighboring residues and the newly identified amino acids in the same domain of the enzyme, and to test the hypothesis that these surrounding residues may play important roles in regulating the drug-sensitivity of the Na+, K+-ATPase and the binding of substrates and digitalis; Aim 5, to examine the interactive role of residues between other extracellular domains (H1-H2 & H3-H4) and the newly identified domains, and to further explore the three-dimensional nature of the digitalis binding pocket. These studies will provide fundamental information to gain insight into the nature of the digitalis-receptor reaction mechanism. The ultimate goal of the research is to obtain new knowledge that will allow us to elucidate the structure-function relationship of the enzyme for a better understanding of the biological processes mediated by the Na+, K+-ATPase in health and disease.

描述（改编自摘要）：Na+、K+-ATP酶（Na泵）是 洋地黄生物作用的靶受体。 精确的 机制以及氨基酸侧链基团的详细知识 负责洋地黄结合仍然是一个悬而未决的问题。 这 该提案的重点是确定该区域和特定的氨基 参与洋地黄与 Na+、K+-ATP 酶结合的酸是 对于离子传输至关重要。 本研究的策略是使用原生 洋地黄及其衍生物及其互补技术涉及 蛋白质化学和分子生物学提供了直接证据 特异性的、共价结合的中间体（酶-洋地黄复合物），即 普通化学分析无法检测到。 纯化的心脏 Na+、K+-ATP 酶 来自兔子和大鼠的样品将用于亲和标记。 新的 确定的残基将指导定点诱变的设计 研究进一步表征和探索洋地黄结合位点 大鼠 Na+、K+-ATP 酶的三种亚型。 五个具体目标是 建议实现的目标：目标 1，确定结构 两种哇巴因敏感的洋地黄结合口袋的决定因素 （兔）和哇巴因抗性（大鼠）Na+、K+-ATP酶，并测试 假设新发现的药物结合位点是通用的 蛋白质水平的洋地黄结合位点；目标 2，表征 新鉴定的氨基酸位于洋地黄结合位点 分子水平，并进一步检验这些位点的假设 Na+、K+-ATPase 三种亚型的通用药物结合位点； 目标 3，确定新鉴定的氨基酸是否有助于 哇巴因敏感性，并测试新的药物结合位点是否 结构上靠近胞外域中的底物结合位点 Na+、K+、ATP 酶；目标4，探索之间的函数关系 相邻残基和新鉴定的氨基酸在同一 酶的结构域，并检验这些周围的假设 残留物可能在调节药物敏感性中发挥重要作用 Na+、K+-ATP酶以及底物与洋地黄的结合；目标5， 检查其他细胞外结构域之间残基的相互作用 (H1-H2 & H3-H4) 和新确定的域，并进一步探索 洋地黄结合口袋的三维性质。 这些研究 将提供基本信息以深入了解事物的本质 洋地黄受体反应机制。 研究的最终目标是 获得新的知识，使我们能够阐明 酶的结构与功能关系，以便更好地理解 健康中由 Na+、K+-ATP 酶介导的生物过程 疾病。