IMMUNOLOGICAL STUDIES OF THE DIGITALIS RECEPTOR

洋地黄受体的免疫学研究

• 批准号: 3343551
• 负责人: William James Ball
• 金额: $ 17.67万
• 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/3343551
• 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，