Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA

FEBS Letters

Schoner, W.¹  Antolovic, R.¹  Horisberger, J.-D.²  Canessa, C.²  Rossier, B.C.²  Geering, K.² 

[1] Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität, Frankfurterstr. D-35392 Geissen, Germany;Institut de Pharmacologie et de Toxicologie de l'Université, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland

关键词: Na;    K-ATPase;    Cardiac steroid;    Xenopus laevis oocyte;    Site-directed mutagenesis;    Affinity labeling;    HDMA;    N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-ϵ-aminocaproate;    Enzyme: Na+/K+-ATPase;    Na+/K+-transporting ATPase (EC 3.6.1.37);   

DOI  :  10.1016/0014-5793(95)00637-O

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonylϵ-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase ϵ 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.

【 授权许可】

Unknown