STRUCTURE/FUNCTION STUDIES OF NA+/K+ ATPASE

NA /K ATP酶的结构/功能研究

• 批准号: 2222949
• 负责人: Elmer M Price
• 金额: $ 16.64万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222949
• 关键词: aminoacid; cardiac glycosides; chemical binding; chemical substitution; enzyme structure; membrane transport proteins; mutant; potassium; site directed mutagenesis; sodium; sodium potassium exchanging ATPase; stoichiometry; tissue /cell culture

Na,K-ATPase is an ubiquitous plasma membrane-derived enzyme which establishes and maintains the electrochemical gradient of Na and K ions across the membranes of most animal cells. This enzyme is also the target enzyme for a class of drugs known as cardiac glycosides, which are used to treat congestive heart failure. Despite the widespread use of these drugs, little is known about the nature of the binding site on the enzyme. The long-term goal of this research is the detailed analysis of the cardiac glycoside binding site on Na,K-ATPase, as well as the characterization of the membrane organization of the enzyme's alpha subunit. The aim of identifying intracellular versus extracellular regions will aid in the generation of a model of the entire enzyme, as well as of the cardiac glycoside binding site, which is known to be extracellular. The approach will utilize a combination of biochemical, immunological and molecular biological methodologies. All of the aims of the proposal entail the use of in vitro mutagenesis to introduce amino acid substitutions in the a subunit of the Na,K-ATPase. These mutants will be expressed in a culture system that has been devised to facilitate the selection of only those mutants that are still biologically active. One aspect of the proposal entails the use of site-directed and random mutagenesis to identify specific amino acids of the Na,K-ATPase alpha subunit that are involved in cardiac glycoside binding. Mutants will be made and transfected into HeLa cells. Cells will be selected in ouabain and if a mutation has been made that interferes with binding, then resistant cells will be generated. In the. case of random mutagenesis, the polymerase chain reaction will be used to identify the amino acid change that mediates the resistant phenotype. In addition, existing ouabain-resistant site-directed mutants, as well as those identified in this project, will be characterized in detail regarding the inhibition potency of several different glycoside analogues and aglycones in an effort to provide insight into what specific regions of Na,K-ATPase interact with particular structural features of the inhibitor molecule. Finally, mutagenesis will be used to introduce an 8 amino acid "flag" sequence into various regions of the alpha subunit. The flag sequence is not only an epitope for a monoclonal antibody, but is a unique specific protease site as well. The flagged alpha subunit will be expressed in HeLa cells. Following the selection of biologically active mutants, intact or permeabilized cells will be incubated with either the monoclonal antibody or the protease. Immunofluorescence or protein immunoblots will be used to determine it the protein reagent was able to interact with its target sequence in intact versus permeabilized cells. Mutagenesis will also be used to study the spatial organization of the extracellular domains of the alpha subunit.

Na，K-ATPase是一种普遍存在的质膜衍生酶 建立和维持Na和K离子的电化学梯度 在大多数动物细胞的细胞膜上。这种酶也是 一种被称为心脏糖苷类药物的靶酶，这类药物是 用于治疗充血性心力衰竭。尽管广泛使用了 对于这些药物，人们对其结合部位的性质知之甚少 酵素。 这项研究的长期目标是详细分析 Na，K-ATPase上的心脏糖苷结合位点，以及 酶α的膜组织结构的表征 亚单位。识别细胞内和细胞外的目的 区域将有助于生成整个酶的模型，因为 以及心脏糖苷结合部位，这是已知的 细胞外。该方法将利用生化、 免疫学和分子生物学方法。的所有目标 这项提议需要使用体外诱变来引入氨基 Na，K-ATPase a亚基的酸取代。这些变种人 将在一种文化系统中表达出来，这种文化系统旨在促进 只选择那些仍然具有生物活性的突变体。 该提案的一个方面需要使用现场定向和随机 突变鉴定Na，K-ATPaseα的特定氨基酸 参与心脏糖苷结合的亚基。变种人将会是 并将其导入HeLa细胞。将在哇巴因中选择单元格 如果发生了干扰结合的突变，那么 就会产生抗性细胞。在。随机突变的情况下， 将使用聚合酶链式反应来鉴定氨基酸 介导抗性表型的变化。此外，现有的 哇巴因抗性定点突变体，以及在 这个项目，将详细描述关于抑制 几种不同的糖苷类似物和苷元在小鼠体内的药效 努力洞察Na，K-ATPase的哪些特定区域 与抑制剂分子的特殊结构特征相互作用。 最后，突变将被用来引入一个8个氨基酸的“旗帜”。 测序到阿尔法亚单位的不同区域。该标志序列是 不仅是单抗的表位，而且是唯一的特异性 还有蛋白水解酶的部位。标记的阿尔法亚单位将以 HeLa细胞。在选择了具有生物活性的突变体之后， 完整或通透性的细胞将与 单抗或蛋白水解酶。免疫荧光或蛋白质 将使用免疫印迹来确定蛋白质试剂是否能够 与其靶序列在完整细胞中相互作用，而不是在通透性细胞中。 诱变技术也将被用来研究 阿尔法亚单位的胞外区。