A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (Pi + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, anidn hibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and bindingis prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-foldf aster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes
