The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.
The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.
Amler
E
E
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Ohio 45267-0575, USA.
Abbott
A
A
Malak
H
H
Lakowicz
J
J
Ball
W J
WJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000873:Anthracenes; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002241:Carbohydrates / Q000737:chemistry; D002301:Cardiac Glycosides / Q000378:metabolism; D002412:Cations; D004735:Energy Transfer; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007546:Isoquinolines; D007700:Kinetics; D008954:Models, Biological; D009038:Motion; D009840:Oligomycins; D010042:Ouabain / Q000031:analogs & derivatives; D011487:Protein Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents
D000873:Anthracenes; D002241:Carbohydrates; D002301:Cardiac Glycosides; D002412:Cations; D005456:Fluorescent Dyes; D007546:Isoquinolines; D009840:Oligomycins; D012997:Solvents; D010042:Ouabain; C013814:anthroylouabain; D000255:Adenosine Triphosphate; C017475:lucifer yellow; D000254:Sodium-Potassium-Exchanging ATPase