Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements

Blanche Schwappach, Werner Stürmer, Hans Jürgen Apell, Steven J D Karlish

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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 (P(i) + 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, 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 dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster 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.
    Original languageEnglish
    Pages (from-to)21620-21626
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume269
    Issue number34
    Publication statusPublished - 26 Aug 1994

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