Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase
- 16 December 1986
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 25 (25) , 8133-8141
- https://doi.org/10.1021/bi00373a002
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 probe directed to specific sites on the .alpha.- and and .beta.-subunits and ligand-induced changes in the fluorescence of a probe specifically attahed 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., and 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 wa 47 .ANG. 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 .ANG.) when anthroylouabain was bound with ATP or Pi as phosphroylating ligands but increased to 49 .ANG. 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. Conformational changes induced by ions, substrates, or inhibitors of the enzyme were not detectable by changes in the spectroscopic parameters of lucifer yellow attached to galactose residues of the .beta.-subunit. Ligands that cause two-dimensional crystallization of (Na,K)-ATPase quenched the fluorescence of lucifer yellow, suggesting that crystallization involves extensive interactions between .beta.-subunits.This publication has 39 references indexed in Scilit:
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