The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

Abstract

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.

Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

“…It had been found previously by energy transfer measurements that ATP and cardiac glycoside binding sites are 7.2 nm apart (57) and that the cytosolic part carrying the ATP site sits 3 nm above the inner side of the plasma membrane (57,58). Additionally, energy transfer measurements between the AOcontaining ouabain binding site and the lucifer yellow-modified ␤-subunit (51,59) give an overall arrangement of distances (Fig. 7).…”

Preparation of Na,K-ATPase Specifically Modified on the Anti-fluorescein Antibody-Inaccessible Site by Fluorescein 5′-Isothiocyanate

“…However, only gel filtration in the presence of anti-fluorescein antibody was effective in removing antibody-accessible, conformation-insensitive fluorescein. The yield of specifically modified enzyme was not reported, but apparently purification of labeled enzyme by gel filtration is impractical because subsequent studies in which ErITC was used as the acceptor for FRET measurements were carried out with heterogeneously labeled enzyme (37). The alternative method of obtaining labeling only on the antibody-inaccessible site reporting the conformational change (Fig.…”

“…However, the latter approach does not work with all fluorescent isothiocyanate probes. For example, antibody directed against ErITC did not quench the fluorescence of ErITC (9), complicating the use of ErITC in FRET experiments (37). Therefore, methods of avoiding or removing nonspecific labeling are important if fluorescent isothiocyanate derivatives are to be used as reporter groups or as donor or acceptor in FRET measurements.…”

The isolated H4‐H5 cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

“…Na,K‐ATPase (EC 3.6.1.3) is a plasma membrane protein consisting of at least two major subunits: the catalytic α‐subunit, with molecular mass about 112 000 and responsible for all currently known transport and catalytic functions, and the associated glycoprotein β‐subunit (molecular mass about 35 000 excluding oligosaccharides) [1]. There is now common agreement that an α 2 β 2 heterodimer forms in the plasma membrane to create the native structure of the enzyme [2, 3]. The α‐subunit contains 10 (or eight) transmembrane segments with a large cytoplasmic loop, located between helices H 4 and H 5 , where the high‐affinity ATP binding site and phosphorylation site are localised [4].…”