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]-(3 beta,5 beta,14 beta, 20E)-24-azido-3-[(2,6-dideoxy-beta-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 degrees C] in the dark with an equilibrium dissociation constant of (1.4 +/- 0.3) X 10(-7) M. Ultraviolet irradiation of a solution of enzyme plus 3H-labeled probe, followed by analysis of covalently incorporated radiolabel, shows ouabain-displaceable labeling exclusively of the alpha 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 alpha subunit of this enzyme.
D000345:Affinity Labels / Q000138:chemical synthesis; D000818:Animals; D001386:Azides / Q000138:chemical synthesis; D001665:Binding Sites; D002301:Cardiac Glycosides / Q000138:chemical synthesis; D010777:Photochemistry; D000254:Sodium-Potassium-Exchanging ATPase; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D002301:Cardiac Glycosides; C040336:24-azido-3-((2,6-dideoxy-beta-hexapyranosyl)oxy)-14-hydroxy-21-norchol-20(22)-en-23-one; D000254:Sodium-Potassium-Exchanging ATPase