STRUCTURE/FUNCTION STUDIES OF NA,K-ATPASE

NA,K-ATP酶的结构/功能研究

• 批准号: 3365611
• 负责人: Elmer M Price
• 金额: $ 17.4万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-10 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3365611
• 关键词: adenosinetriphosphatase; aminoacid; cardiac glycosides; chemical binding; chemical substitution; enzyme structure; membrane transport proteins; mutant; potassium; site directed mutagenesis; sodium; 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-ATP酶是一种普遍存在的质膜衍生酶， 建立并维持 Na 和 K 离子的电化学梯度 可以穿过大多数动物细胞的细胞膜。 这种酶也是 一类被称为强心苷的药物的靶酶，这些药物是 用于治疗充血性心力衰竭。 尽管广泛使用 对于这些药物的结合位点的性质知之甚少 酶。 本研究的长期目标是详细分析 Na,K-ATP酶上的强心苷结合位点，以及 酶α膜组织的表征 亚基。 识别细胞内与细胞外的目的 区域将有助于生成整个酶的模型，如 以及强心苷结合位点，已知是 细胞外的。 该方法将结合生物化学、 免疫学和分子生物学方法。 所有的目标 该提案需要使用体外诱变来引入氨基 Na,K-ATP 酶 a 亚基中的酸取代。 这些突变体 将在一个文化系统中表达出来，该系统旨在促进 仅选择那些仍具有生物活性的突变体。 该提案的一方面需要使用定点和随机 诱变以鉴定 Na,K-ATPase α 的特定氨基酸 参与强心苷结合的亚基。 突变体将是 制备并转染到 HeLa 细胞中。 将在哇巴因中选择细胞 如果发生了干扰结合的突变，那么 就会产生耐药细胞。 在.随机突变的情况下， 聚合酶链式反应将用于鉴定氨基酸 介导耐药表型的变化。 此外，现有 哇巴因抗性定点突变体，以及那些在 该项目将详细描述有关抑制的特征 几种不同的糖苷类似物和糖苷配基的效力 努力深入了解 Na,K-ATP 酶的特定区域 与抑制剂分子的特定结构特征相互作用。 最后，将使用诱变引入 8 个氨基酸“标志” 序列到α亚基的各个区域。 标志序列是 不仅是单克隆抗体的表位，而且是独特的特异性 蛋白酶位点也是如此。 标记的 alpha 亚基将表示为 海拉细胞。 选择具有生物活性的突变体后， 完整的或透化的细胞将与 单克隆抗体或蛋白酶。免疫荧光或蛋白质 免疫印迹将用于确定蛋白质试剂能够 在完整细胞和透化细胞中与其靶序列相互作用。 诱变也将用于研究空间组织 α 亚基的细胞外结构域。