Structure-Function Relationships in Na, K-ATPase

这项研究的长期目标是了解ATP如何通过Na， k - ATP酶驱动离子运输。大量证据支持ATP水解通过底物和离子诱导的构象转变与转运步骤相关的机制。尽管我们对Na， k - atp酶的酶学性质已经有了相当详细的了解，但对Na， k - atp酶的主要E1和E2状态的结构知之甚少，对其提出的亚构象就更少了。该项目包括旨在进一步描绘酶的构象状态的实验，特别是相互转化以及它们之间的结构和功能关系。目前的研究在结构和动力学研究的五个主要领域得到了继续和扩展。(1)利用福斯特能量转移测量的距离确定构象选择性处理形成的酶态之间的结构关系。(2)荧光停流法测定反应周期中构象步骤的速率。(3)用荧光碳二亚胺标记假定的离子结合结构域。(4)利用纯化肽的抗体测定IAF结合区的功能。(5)利用蛋白测序技术研究Na， k - atp酶荧光标记的物种差异。离子跨膜运输是由特定的运输系统精确调节的。有一种“泵”蛋白在细胞膜上交换钠离子和钾离子。这在调节细胞体积、酸度和离子组成方面起着重要作用。众所周知，泵蛋白在离子运输过程中会改变形状。在这个过程中，细胞保存能量是很重要的。该项目将提供有关泵结构及其参与能量代谢的新信息。* * *​​​​​​ //

The long-term goal of this research is to understand how ATP drives ion transport via the Na,K-Atpase. A great deal of evidence supports a mechanism in which ATP hydrolysis is linked to the transport steps through conformational transitions induced by substrate and ions. Despite the fact that the enzymatic properties are known in considerable detail, little is known about the structures of the major E1 and E2 states of the Na,K-ATPase, and even less about proposed subconformations. This project consists of experiments designed to delineate further the conformational states of the enzyme, in particular the interconversions and the structural and functional relationships among them. Current studies are continued and extended in five major areas of structural and kinetic investigations. (1) Determination of structural relationships among enzyme states formed by conformationally selective treatments using distances measured by Forster energy transfer. (2) Measurement of rates of the conformational steps in the reaction cycle with fluorescence stopped-flow. (3) Labeling of the putative ion binding domain with fluorescent carbodiimides. (4) Determination of the function of the IAF binding region using antibodies raised to a purified peptide. (5) Investigation of species differences in fluorescent labeling of Na,K-ATPase by protein sequencing techniques. Ion transport across membranes is precisely regulated by specific transport systems. There is a "pump" protein which exchanges sodium ions and potassium ions across membranes. This plays an important role in regulating cell volume and acidity and ion composition. It is known that the pump protein changes shape during ion transport. It is important to the cell to conserve energy during this process. This project will provide new information on the structure of the pump and its involvement in energy metabolism.*** //