INTERACTION OF DIGITALIS WITH ITS RECEPTOR NA+/K+ ATPASE

本建议的长期目标是确定分子 洋地黄类药物（强心苷）的详细信息 与其药理学受体（Na，K）-ATP酶相互作用。到 了解这个过程，受体和药物的结构， 这些结构的变化对稳定性（亲和力）的影响 必须测定药物-受体复合物。人的心脏包含三个 同种型，α 1在肌肉中占优势，α 2和α 3在肌肉中占优势， 丰富了指挥系统。如果α 1亚型主要是 负责收缩性和α 2和/或α 3的增加 主要负责毒性作用的强心苷， 对α 1的亲和力相对较高（因此具有 改善的治疗指数）。具体 目的1.鉴定人（Na，K）-ATP酶的三种α同工酶 在NIH 3 T3细胞中表达的差异相互作用 一系列强心苷具体目标2是确定和 位于一级序列内，（Na，K）- 参与与强心苷相互作用的ATP酶。网站 感兴趣的包括配体调节位点（Mg、ATP、Na和K）以及 实际的强心苷结合位点。（Na，K）-ATP酶的结构 将使用绵羊α 1同种型的定点突变来改变 在NIH 3 T3或Hela细胞中表达。药物的结构将是 通过利用现有的洋地黄类似物来改变。目标1和 2解离常数KD将通过饱和结合来确定。 测定法或通过测量动力学速率常数， 从抑制研究中推断（150）。结合的KD的改变 一系列强心苷的作用将决定 特定的氨基酸突变在结合过程中起作用。具体目标3 是获得和表征α-β复合物的制剂， 分离α和β亚基并选择较小的蛋白质片段 适当纯度的绵羊（Na，K）-ATP酶， 三维结构研究 将使用以下方法评估纯度 天然和变性凝胶以及使用抗体开发的蛋白质印迹 对抗绵羊的α和β亚基 糖基化程度， 二硫键和二级结构（环状）的数量和完整性 二向色性和红外光谱）将被测定。一个工作 受体的实验室模型与实验数据一致 将使用能量最小化技术来构建。蛋白质， 当纯的时候，将被提交用于结晶试验。

The long term objective of this proposal is determination of the molecular details by which the drugs of the digitalis class (cardiac glycosides) interact with their pharmacological receptor, the (Na,K)-ATPase. To understand this process, the structure of the receptor and the drug, and the effect of changes in these structures on the stability (affinity) of the drug-receptor complex must be determined. Human heart contains three isoforms, alpha1 being predominant in muscle and alpha2 and alpha3 being enriched in the conducting system. If the alpha1 isoform is primarily responsible for the increase in contractility and alpha2 and/or alpha3 primarily responsible for the toxic effect, a cardiac glycoside having a relatively higher affinity for alpha1 (and therefore possessing an improved therapeutic index) might be discovered or synthesized. Specific Aim 1 is to characterize the three alpha isozymes of human (Na,K)-ATPase expressed in NIH 3T3 cells with respect to their differential interaction with a series of cardiac glycosides. Specific Aim 2 is to identify and locate within the primary sequence, amino acid residues in the (Na,K)- ATPase that are involved in interacting with cardiac glycosides. The sites of interest include ligand regulatory sites (Mg, ATP, Na and K) as well as the actual cardiac glycoside binding site. The structure of (Na,K)-ATPase will be altered using site-directed mutagenesis of sheep alpha1 isoform expressed in NIH 3T3 or in Hela cells. The structure of the drug will be varied by taking advantage of existing digitalis analogous. For Aims 1 and 2 the dissociation constant, KD, will be determined by saturation binding assays or by measurement of kinetic rate constants or alternatively, deduced from inhibition studies (150). The alteration in KD for binding of a series of cardiac glycosides to each mutant will define the role specific amino acids mutated play in the binding process. Specific Aim 3 is to obtain and characterize preparations of the alpha-beta complex, separated alpha and beta subunits and selected smaller protein fragments of sheep (Na,K)-ATPase of appropriate purity and in sufficient amounts for three dimensional structural studies. Purity will be assessed using native and denaturing gels and Western blots developed using antibodies against the sheep alpha and beta subunits. Degree of glycosylation, number and intactness of disulfide bonds and secondary structure (circular dichroism and infrared spectroscopy) will be determined. A working laboratory model of the receptor consistent with the experimental data will be constructed using techniques of energy minimization. The proteins, when pure, will be submitted for crystallization trials.