Abstract
(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)
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