lucifer-yellow and anthroylouabain

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.

Topics: Adenosine Triphosphate; Animals; Anthracenes; Binding Sites; Biophysical Phenomena; Biophysics; Carbohydrates; Cardiac Glycosides; Cations; Energy Transfer; Fluorescent Dyes; In Vitro Techniques; Isoquinolines; Kinetics; Models, Biological; Motion; Oligomycins; Ouabain; Protein Conformation; Sheep; Sodium-Potassium-Exchanging ATPase; Solvents

(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)

Topics: Animals; Anthracenes; Binding Sites; Cardiac Glycosides; Cell Membrane; Dogs; Glycoproteins; Isoquinolines; Kidney; Kinetics; Macromolecular Substances; Oligosaccharides; Ouabain; Protein Binding; Sodium-Potassium-Exchanging ATPase; Spectrophotometry

Sodium plus potassium activated adenosinetriphosphatase [(Na,K)ATPase] is composed of a catalytic subunit (alpha) and a glycoprotein subunit (beta) of unknown function. A method has been developed to label the beta subunit of purified dog kidney (Na,K)ATPase with fluorescent probes. The method consists of oxidation of beta-subunit oligosaccharides, reaction of the resulting aldehydes with fluorescent hydrazides, and reduction of the hydrazones and unreacted aldehydes with NaBH4. Two oxidation methods were compared. Simultaneous treatment with neuraminidase and galactose oxidase did not inhibit significantly (Na,K)ATPase activity and allowed insertion of up to 11 mol of probe per mol of beta. In contrast, oxidation of (Na,K)ATPase oligosaccharides with periodate resulted in 50-80% inhibition of the (Na,K)ATPase activity with low or undetectable labeling. Eleven commercial probes and two novel hydrazides were tested for labeling of (Na,K)ATPase treated with galactose oxidase and neuraminidase. Eight probes did not label (Na,-K)ATPase but labeled red cell ghosts oxidized with periodate. Four probes labeled beta specifically but either adsorbed to the membrane tightly, or cross-linked the beta subunits, or formed unstable adducts. Lucifer yellow CH labeled beta specifically without membrane adsorption. Labeling stoichiometries from 1 to 11 mol of lucifer yellow CH per mol of beta were obtained without inhibition of (Na,K)ATPase activity and without significant alteration of the anthroylouabain binding capacity or its association and dissociation kinetics. Anthroylouabain specifically bound to the lucifer-labeled (Na,K)ATPase had a decreased quantum yield, probably due to resonance energy transfer. This suggests that the sites of lucifer attachment on beta are within energy transfer distance from the cardiac glycoside site on alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anthracenes; Dogs; Fluorescein; Fluoresceins; Galactose Oxidase; Glycoproteins; Hydrazines; Isoquinolines; Kidney; Macromolecular Substances; Neuraminidase; Ouabain; Oxidation-Reduction; Sodium-Potassium-Exchanging ATPase