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 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.
Schwappach
B
B
Department of Biology, University of Konstanz, Federal Republic of Germany.
Stürmer
W
W
Apell
H J
HJ
Karlish
S J
SJ
D000818:Animals; D002301:Cardiac Glycosides / Q000378:metabolism*; D002414:Cations, Monovalent / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004560:Electricity; D005456:Fluorescent Dyes / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism; D008954:Models, Biological; D009038:Motion; D010446:Peptide Fragments / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D011817:Rabbits; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013343:Styrenes / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D002301:Cardiac Glycosides; D002414:Cations, Monovalent; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D012964:Sodium; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium