Photoaffinity labeling of the sodium- and potassium-activated adenosinetriphosphatase with a cardiac glycoside containing the photoactive group on the C-17 side chain

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Abstract
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 (Na,Kl-ATPase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl2, 3 mM phosphate, 2 mM EDTA, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37.degree. C] in the dark with a Kd of (1.4 .+-. 0.3) .times. 10-7 M. UV 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 (Na,K)-ATPase. The binding site of the C-17 side group of cardiac glycosides is evidently located on or near the .alpha. subunit of this enzyme. [Digitalis glycosides are widely used in the treatment of cardiac dysrhythmias and congestive heart failure.].

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