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

这项提议的长期目标是确定分子 洋地黄类药物(强心苷)的使用情况 与它们的药理受体(Na，K)-ATPase相互作用。至 了解这一过程，受体和药物的结构，以及 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体。人的心脏包含三个 异构体，α1在肌肉中占主导地位，α2和α3是 丰富的指挥系统。如果α1亚型主要是 对收缩能力和α2和/或α3的增加负责 对这种毒性作用负有主要责任的是一种具有 对Alpha1的亲和力相对较高(因此拥有 改善的治疗指数)可能被发现或合成。特定的 目的1研究人(Na，K)-ATPase的三种α-同工酶 在NIH 3T3细胞中的表达与它们的差异相互作用 含有一系列的强心苷。具体目标2是确定和 位于一级序列内，氨基酸残基位于(Na，K)- 参与与心脏糖苷相互作用的ATPase。这些网站 感兴趣的包括配体调节位点(镁、三磷酸腺苷、钠和钾)以及 实际的心脏糖苷结合部位。(Na，K)-ATPase的结构 将通过对绵羊α1亚型进行定点突变来改变 在NIH3T3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而变化。对于AIMS 1和 解离常数Kd将由饱和结合来确定 化验或通过测量动力学速率常数或可替换地， 从抑制研究中得出(150)。Kd中对结合的改变 每个突变体的一系列心脏糖苷将定义其作用 特定的氨基酸突变在结合过程中发挥作用。具体目标3 是为了获得和表征α-β复合体的制剂， 分离了α和β亚基并选择了较小的蛋白质片段 适当纯度和足够数量的绵羊(Na，K)-ATPase 三维结构研究。纯度将使用以下方法进行评估 利用抗体形成的天然和变性凝胶和蛋白质印迹 对抗绵羊的阿尔法和贝塔亚基。糖基化程度， 二硫键的数目和完整性以及二级结构(圆形 二向色性和红外光谱)将被测定。一名工作人员 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进行建造。这些蛋白质， 提纯后，将提交结晶试验。

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.