The long term objective of this project is to understand the reaction mechanism of the Na,K pump in terms of its quaternary and tertiary structure. In this application we propose to develop a relatively simple method for obtaining large amounts of pure, fully active enzyme from a secretory epithelium; such a method is not currently available. We propose to use this enzyme in a variety of ways. We will attempt to solubilize the enzyme in detergents while maintaining its activity and homogeneity. Such a solubilized enzyme preparation would be suitable for preparing 2D and 3D crystals for structural analysis. We will obtain information about the quaternary structure of the pump in situ by measuring fluorescence energy transfer between fluorescent derivatives of ouabain bound randomly to pumps in membrane bound enzyme preparations and the microsomes from which they are prepared. We will also measure fluorescence energy transfer between pumps of membranes from a variety of other cells with relatively tow pump density. We will use our active enzyme preparation to settle some long standing controversies about the pump reaction mechanism. By preparing enzyme with specific activity close to theoretic maximum, we will demonstrate unambiguously that """"""""half-of'sites"""""""" or """"""""silent partner"""""""" mechanisms are not possible. We will perform experiments which should settle the question of whether protomer interaction modifies the reaction mechanism, and we will re- evaluate some reported findings which have been interpreted in terms of pump structure. We will perform experiments to accurately determine, with a fully active preparation, the number of binding sites for the principal pump ligands, Na,K and ATP, present in each functional pump unit. Because of the high specific activity of our preparation, we will be able to resolve the debate about whether high affinity binding sites for Na coexist with Rb occlusion sites. Such information is crucial for the interpretation of point mutation studies and chemical modification studies. The Na,K pump is the source of energy for many biological processes including cell volume control, communication by means of action potentials, solute absorption and secretion (it provides the energy for the chloride flux carried out by the Cl channel which is defective in cystic fibrosis), etc. The Na,K pump is the receptor for the cardioactive steroids used in the treatment of congestive heart failure, and it has been implicated in the pathogenesis of essential hypertension. Knowledge of its structure and function would improve our understanding of these processes, and would improve our ability to design drugs which might beneficially modify behavior of the pump in clinical situations.
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