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

Tamboue Deffo, Dwight S. Fullerton, Masaru Kihara, Reginald H. McParland, Robert R. Becker, Michael I. Schimerlik, Arthur H From, Khalil Ahmed

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)6303-6309
Number of pages7
JournalBiochemistry
Volume22
Issue number26
DOIs
StatePublished - 1983

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