Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase.
(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000873:Anthracenes / Q000378:metabolism*; D001665:Binding Sites; D002301:Cardiac Glycosides / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004285:Dogs; D006023:Glycoproteins / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D009844:Oligosaccharides / Q000032:analysis*; D010042:Ouabain / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013053:Spectrophotometry
D000873:Anthracenes; D002301:Cardiac Glycosides; D006023:Glycoproteins; D007546:Isoquinolines; D046911:Macromolecular Substances; D009844:Oligosaccharides; D010042:Ouabain; C013814:anthroylouabain; C017475:lucifer yellow; D000254:Sodium-Potassium-Exchanging ATPase