IMMUNOLOGICAL STUDIES OF THE DIGITALIS RECEPTOR

这个项目的目标是确定结构， 膜结合的Na~+，K~+-的组织和功能 ATPase。这种酶对和的活性调节是必不可少的 维持细胞内的Na+和K+水平。它也是世界上 心脏糖苷的药理受体，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 对酶活性的抑制以及这种抑制如何与 这种药物对心肌收缩有变力作用。在这 我们将继续研究其作用机制 单抗对酶功能的影响 确定它们是如何抑制酶活性并改变正常的 与之结合的调节配体之间的相互作用 Na+，K+-ATPase的催化或α亚基。这是 通过研究Na+，K+-ATPase，p- 硝基苯基磷酸酶和乙酰磷酸酶活性，以及 磷酸酶中间体的形成和部分反应 去磷酸化。配体诱导的细胞内蛋白质构象变化 使用荧光探针标记的酶来监测酶。 这些研究将提供有关心脏疾病的新信息 糖苷作用。此外，我们还将使用合成肽， 有酶不同区域的氨基酸序列 鉴定该酶的抗原性部位。多克隆抗体 产生了这些多肽和全酶导向的抗体 结合这些多肽的哪些将被用来定位功能 心脏糖苷、三磷酸腺苷和阳离子结合等区域 酶的位置。我们的全酶和合成产品系列 多肽导向的抗体也将被用于探测第三代 酶的结构。最后，我们将使用各种 免疫化学技术确定这两种组织 和可能的功能作用，β，糖蛋白亚基 Na+，K+-ATPase。然后我们将确定它的机制(S)。 对酶催化活性的影响。

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.