IMMUNOLOGICAL STUDIES OF THE DIGITALIS RECEPTOR

洋地黄受体的免疫学研究

• 批准号: 3343550
• 负责人: William James Ball
• 金额: $ 17.22万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3343550
• 关键词: adenosine triphosphate; adenosinetriphosphatase; antibody; antigens; binding proteins; chemical structure function; digitalis; drug receptors; enzyme mechanism; enzyme structure; fluorescence spectrometry; fluorescent dye /probe; genetic translation; immunochemistry; immunopharmacology; laboratory mouse; laboratory rabbit; monoclonal antibody; ouabain; peptide chemical synthesis; phosphorylation; protein engineering; sodium potassium exchanging ATPase

The goal of this project is to determine the structure, organization and function of the membrane bound Na+, K+- ATPase. This enzyme is essential for the active regulation of and maintenance of Na+ and K+ levels within the cell. It is also the pharmacological receptor for cardiac glycosides which are used to treat some heart diseases. These studies are designed to help elucidate the molecular mechanisms of cardiac glycoside inhibition of enzyme activity and how this inhibition is linked to the drug's inotropic effects on heart muscle contraction. In this project we will continue with studies of the mechanisms of monoclonal antibody effects on enzyme function in order to determine how they inhibit enzyme activity and alter the normal interactions between the regulatory ligands which bind to the catalytic, or alpha subunit of Na+, K+-ATPase. This is accomplished by studying the Na+,K+-ATPase, p- nitrophenylphosphatase, and acetylphosphatase activities, and the partial reactions of phosphoenzyme intermediate formation and dephosphorylation. Ligand-induced conformational changes in the enzyme are monitored using fluorescent probe-labeled enzyme. These studies will provide new information about cardiac glycoside action. In addition we will use synthetic peptides which have the amino acid sequences of various regions of the enzyme to identify antigenic sites of the enzyme. Polyclonal antibodies raised to these peptides and holoenzyme-directed antibodies which bind to these peptides will be used to locate functional regions such as the cardiac glycoside, the ATP and cation binding sites of the enzyme. Our collection of holoenzyme- and synthetic peptide-directed antibodies will also be used to probe the tertiary structure of the enzyme. Finally, we will use various immunochemical techniques to determine both the organization and possible functional role of beta, the glycoprotein subunit of Na+, K+ -ATPase. We will then determine the mechanism(s) of its effects on enzyme catalytic activity.

这个项目的目标是确定结构， 膜结合Na+，K+-的组织和功能 ATP酶 这种酶是必不可少的积极调节和 维持细胞内的Na+和K+水平。 也是 强心苷的药理学受体，其用于 治疗一些心脏病。 这些研究旨在帮助 阐明强心苷的分子机制 酶活性的抑制以及这种抑制如何与 这种药物对心肌收缩的正性肌力作用。 在这 项目我们将继续研究的机制， 单克隆抗体对酶功能的影响， 确定它们如何抑制酶的活性， 结合到细胞的调节配体之间的相互作用 Na+，K+-ATP酶的α亚基。 这是 通过研究Na+，K+-ATP酶，p- 硝基苯基磷酸酶和乙酰磷酸酶活性，以及 磷酸酶中间体形成的部分反应， 去磷酸化 配体诱导的构象变化 使用荧光探针标记的酶监测酶。 这些研究将为心脏病的治疗提供新的信息。 糖苷作用 此外，我们将使用合成肽， 具有酶的各个区域的氨基酸序列 来鉴定酶的抗原位点。 多克隆抗体 这些肽和全酶导向的抗体 结合到这些肽上的抗体将用于定位功能性的 强心苷、ATP和阳离子结合区 酶的位置。 我们收集的全酶和合成酶 肽定向抗体也将用于探测三级结构， 酶的结构。 最后，我们将使用各种 免疫化学技术来确定组织 和β的可能的功能作用，β的糖蛋白亚基， Na+，K+ -ATP酶。 然后我们将确定其机制（S） 对酶催化活性的影响。