Photoaffinity Labeling of the Sodium- and Potassium-Activated Adenosinetriphosphatase with a Cardiac Glycoside Containing the Photoactive Group on the C-17 Side Chain

The synthesis and properties of a radiolabeled glycoside photoaffinity probe, [³H]-(3β,5β,14β,20E)-24-azido-3-[(2,6-dideoxy-β-d-ribo-hexopyranosyl)oxy]-14-hydroxy-21-norchol-20(22)-en-23-one, containing the photoactive group at the C-17 side chain of the steroid moiety are reported. The molecule binds to the sodium- and potassium-activated adenosinetriphosphatase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl₂, 3 mM phosphate, 2 mM ethylenediaminetetraacetic acid, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37°C] in the dark with an equilibrium dissociation constant of (1.4 ± 0.3) × 10⁻⁷ M. Ultraviolet irradiation of a solution of enzyme plus ³H-labeled probe, followed by analysis of covalently incorporated radio-label, shows ouabain-displaceable labeling exclusively of the α subunit of the sodium- and potassium-activated adenosinetriphosphatase. These data indicate that the binding site of the C-17 side group of cardiac glycosides is located on or near the α subunit of this enzyme.