TY - JOUR
T1 - Photoaffinity Labeling of the Sodium- and Potassium-Activated Adenosinetriphosphatase with a Cardiac Glycoside Containing the Photoactive Group on the C-17 Side Chain
AU - Deffo, Tamboue
AU - Fullerton, Dwight S.
AU - Kihara, Masaru
AU - McParland, Reginald H.
AU - Becker, Robert R.
AU - Schimerlik, Michael I.
AU - From, Arthur H
AU - Ahmed, Khalil
PY - 1983
Y1 - 1983
N2 - 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.
AB - 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.
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U2 - 10.1021/bi00295a041
DO - 10.1021/bi00295a041
M3 - Article
C2 - 6318813
AN - SCOPUS:0021109010
SN - 0006-2960
VL - 22
SP - 6303
EP - 6309
JO - Biochemistry
JF - Biochemistry
IS - 26
ER -