Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.
The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.
Antolovic
R
R
Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität, Giessen, Germany.
Schoner
W
W
Geering
K
K
Canessa
C
C
Rossier
B C
BC
Horisberger
J D
JD
D000345:Affinity Labels; D000818:Animals; D003545:Cysteine / Q000032:analysis*; D004076:Digoxigenin / Q000031:analogs & derivatives*; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013388:Succinimides; D014981:Xenopus
D000345:Affinity Labels; D011994:Recombinant Proteins; D013388:Succinimides; C073892:N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D004076:Digoxigenin