The purpose of this research is to characterize quantitatively how the physiological ligands of the sodium pump regulate active transport in human red cells. Current ideas on the reaction pathway by which the cardiac glycoside-sensitive Na pump catalyzes the exchange of Na and K are derived from experimental data from two sources, enzymatic studies where vectorial and transport properties are lost and transport studies in intact systems, e.g. red cells, where measurement of enzymatic conversions have been difficult. In the present work resealed human red cell ghosts will be used where independent control of intracellular and extracellulare cations is possible. A new procedure for ghost preparation, involving cell lysis during gel filtration, will be used to produce ghosts with little contamination by cytosolic enzymes. 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 caged-ATP or caged-Pi we will study the cation requirements for ATP:ADP exchange and for other partial activities and the effects of Na,K and their cogeners on membrane phosphorylation and dephosphorylation. Using this procedure we will examine in detail the coupling between Na:Na exchange and ATP:ADP exchange in resealed ghosts, reactions involved in Na binding and release. Studies will also be performed on the regulation of Na pump transport modes by Mg and the effect of intracellular pH on cation affinities. The overall aim is to determine to what extent existing models of the Na pump account for the physiologic functioning of Na:K exchange. Recent experimental evidence suggests an abnormal working of the red cell Na pump on a variety of disease states, e.g. hypertension, obesity, muscular dystrophy, sickle cell anemia. A more detailed understanding of the functioning of the normal Na pump is required before these observations can be properly evaluated in diseased states.
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