The long term objective of this project is to understand the reaction mechanism of the Na, K pump. We have developed a procedure for preparing homogeneous, fully active membrane bound Na, K ATPase from nasal glands of salt adapted ducks. We plan to fully characterize the enzyme by determining its primary structure, its phosphorylation capacity from ATP and from inorganic phosphate under a variety of conditions, the characteristics of its interaction with Na and Rb, and its stoichiometry for Rb occlusion. We will determine whether the enzyme is kinetically heterogeneous as are Na, K ATPase preparations from other sources. It is possible that the superior activity of our enzyme is the result of post-translational modification (specifically, regulatory phosphorylation) since the enzyme ia the product of enhanced enzyme synthesis during salt adaptation. This will be addressed by detection of phosphate bound to the enzyme soon after its removal from microsomes. Comparison of the properties of this enzyme with those of partially active renal enzyme should allow a determination of the cause of the reduced activity and the likely consequences of partial activity in functional studies using the enzyme. Theses studies will allow a clearer understanding of nasal gland enzyme and of renal enzyme, and allow a basis for deciding how results obtained with partially active enzyme should be viewed. We will address some questions about the reaction mechanism using our enzyme. Because of its high specific activity, we wil be able to charaterize ion binding with great accuracy There are many reasons to believe that one of the Na ions transported out is different from the other two. We will attempt to bind Na to this site on the outside and learn if it is responsible for modification of the interaction of the enzyme with the inhibitor vanadate, Which forms a transition state analog of one of the enzyme intermediates in the reaction sequence. It is generally believed that ion occlusion is an integral part of the reaction mechanism. As far as we know that belief has never been critically evaluated, and we plan to do so. Comparison of a high resolution structure of the closely related SERCa pump in the Ca bound conformation with a lower resolution structure of vanadate bound nasal gland enzyme suggests to us that the major coriformational change must occur before phosphorylation. If so, this will require a major rethinking about how active tansport occurs, and we will try to identify the location in the reaction mechanism of the conformational change. The Na, K pump is the source of energy for many essential biological processes including cell volume control, communication by means of action potentials, solute reabsorption and secretion, etc. The Na, K pump is the receptor for cardioactive steroids used in the treatment of congestive heart failure, and it has been implicated in the etiology of hypertension. Knowledge of its structure and function would improve our understanding of those processes.
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