NA+/K+ ATPASE--RELATION OF STRUCTURE TO FUNCTION

NAK-ATPase是质膜的内源性酶，维持 大多数藻类表面膜上的正常Na+和K+梯度 真核细胞；是正性变力作用的受体 洋地黄类药物在心肌中的含量。这个项目的长期目标是 项目是定义跨膜离子的分子机制 通过这种酶的运动，并阐明 在生理和病理生理条件下控制其功能 条件。协作建议书的重点是功能 酶的四级结构的意义，并基于 合作调查员的调查结果表明 鸭鼻盐腺酶的亚基相互作用可能有所不同 与那些被广泛使用的酶显著不同 由首席调查员。建议的具体目标 比较研究如下：1.测定各样品的化学计量学 鸭盐腺的活性部位和几个配基结合部位 酶，看看这种制剂是否表现出半部位反应性，并 将这些化学计量学与已知的 肾酶。2.检验平衡结合图的性质 对于几种与鸭盐腺酶相互作用的配体 确定这些是否支持配体的协同结合 该酶的底物-速度曲线表明。3.学习 哇巴因结合鸭的盐腺酶，并比较其效果 该酶的几种生理配体对哇巴因释放的影响 盐腺和肾脏的酶，以表征 两种制剂中配体结合部位的多样性。4.至 研究膜结合型鸭盐腺的亚基关联 通过化学交联剂研究酶。5.获得积极主动和 鸭盐腺酶的稳定洗涤剂增溶制剂 和肾脏的酶，并比较这些的几个性质 与相应的膜结合型制剂的制剂相比较。

NaK-ATPase is the intrinsic enzyme of the plasma membrane that maintains the normal Na+ and K+ gradients across the surface membranes of most eucaryotic cells; and is the receptor for the positive inotropic actions of digitalis drugs in the myocardium. The long-term objectives of this project are to define the molecular mechanisms for the transmembrane ion movements through this enzyme, and to elucidate the mechanisms that control its function under physiological and pathophysiological conditions. The collaborative proposal is focused on the functional significance of the quaternary structure of the enzyme, and is based on the findings of the collaborating investigator suggesting that the subunit interactions of the duck nasal salt gland enzyme may differ significantly from those of the enzymes that have been used extensively by the Principal Investigator. The specific aims of the proposed comparative studies are: 1. To determine the stoichiometries of the active site and several ligand binding sites of the duck salt gland enzyme to see if this preparation exhibits half-site reactivity, and to compare these stoichiometries with the known stoichiometries of the kidney enzyme. 2. To examine the nature of the equilibrium binding plots for several ligands that interact with the duck salt gland enzyme to determine if these support the cooperative binding of ligands as suggested by the substrate-velocity curves of the enzyme. 3. To study ouabain binding to the duck salt gland enzyme, and to compare the effects of several physiological ligands of the enzyme on ouabain release from the salt gland and the kidney enzymes in order to characterize the multiplicity of the ligand binding sites in the two preparations. 4. To examine the subunit associations of the membrane-bound duck salt gland enzyme through chemical cross-linking studies. 5. To obtain active and stable detergent-solubilized preparations of the duck salt gland enzyme and the kidney enzyme, and to compare several properties of these preparations with those of the corresponding membrane-bound preparations.