BINDING OF SODIUM-IONS AND CARDIOTONIC STEROIDS TO NATIVE AND SELECTIVELY TRYPSINIZED NA,K PUMP, DETECTED BY CHARGE MOVEMENTS

A fluorescent dye, RH421, has been used to characterize charge movemen ts associated with cation and cardiotonic steroid binding to Na,K-ATPa se and to a specifically trypsinized preparation, so-called ''19-kDa m embranes.'' A fluorescence decrease induced by Na+ is attributed to el ectrogenic binding of one Na+ ion from the cytoplasm. The apparent aff inity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P- i + Mg)-induced fluorescence signals observed with native enzyme are n ot observed in 19-kDa membranes, consistent with loss of ATP binding a nd phosphorylation. Cardiotonic steroids (CS) bind to native enzyme an d 19-kDa membranes as judged by TH421 signals, fluorescence of anthroy l ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissoc iation are identical in both preparations, by aglycones bind and disso ciated about 6-fold faster to 19-kDa membranes. Binding of Na+ and car diotonic steroids is inactivated upon heating or extensive Pronase dig estion of 19-kDa membranes. This suggests that cation and CS binding d epend on the structural integrity of a complex of the proteolytic frag ments, and that sites for both cations or CS consist of ligating group s located on more than one fragments of 19-kDa membranes.