The purpose of this research is to formulate a molecular mechanism of the action of the sodium pump in cell membranes. Current ideas on the reaction pathway by which the cardiac glycoside-sensitive Na, K-ATPase catalyzes the exchange of Na and K are derived from experimental data from two sources, enzymatic studies in systems where all vectorial and transport properties are lost and transport studies in more intact systems, often human erythrocytes, where the precise measurement of enzymatic steps is difficult. In the present work the properties of human erythrocyte ghosts will be utilized where independent control of intracellular and extracellular compartments is possible. Stable, caged-ATP or other phosphate-containing substrates can be sealed into ghosts when, following a brief pulse of light, free ATP or substrate is released. Using this new photorelease procedure with the caged-ATP or caged Pi it is now feasible to study the cation requirements for ATP:ADP exchange and for other partial enzymatic activities and the effects of Na, K and their cogeners on enzymatic phosphorylation and dephosphorylation. Using this new photorelease procedure it is possible to examine in detail the coupling between Na:Na exchange and ATP:ADP exchange in sealed ghosts, reactions intimately involved in Na binding and release. In studies using purified enzyme from renal medulla, isolated and reconstituted into phospholipid vesicles together with the caged-ATP approach it is possible to perform studies on enzyme conformational changes and transport where the red cell enzyme has too low specific activity to make the experiments possible. Intrinsic protein fluorescence or the fluorsecence of modified enzyme can be monitored following photolytic release of intravesicular pump substrates. The sided effects of cations and the effects of enzyme modification on activity and transport can then be studied in the same preparations. The overall aim is to determine to what extent existing models of the Na pump involving phosphoenzyme intermediates account for tis physiological function of Na, K exchange. Recent experimetal evidence suggests an abnormal working of the red cell Na pump in a variety of disease states, e.g. hypertension, obesity, muscular dystrophy, sickle cell anemia, a more detailed understanding of the funmctioning of the normal Na pump is required before these observations can be properly evaluated in diseased states.
