Antolovic R, Schoner W, Geering K, Canessa C, Rossier B C, Horisberger J D
Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität, Giessen, Germany.
FEBS Lett. 1995 Jul 10;368(1):169-72. doi: 10.1016/0014-5793(95)00637-o.
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.
地高辛配基衍生物N-羟基琥珀酰亚胺地高辛配基-3-O-甲基羰基-ε-氨基己酸酯（HDMA）已被证明可共价标记钠钾ATP酶ε亚基的哇巴因结合位点[安托洛维奇等人（1995年），《欧洲生物化学杂志》227卷，61 - 67页]。在本研究中，我们观察到非洲爪蟾卵母细胞中过表达的野生型钠钾ATP酶的标记和活性失活情况。相比之下，当第一个跨膜片段的半胱氨酸-113突变为丝氨酸时，未检测到明显抑制作用和标记，尽管在急性抑制实验中测得的该突变体对地高辛配基或HDMA的亲和力与野生型相似。这表明在其配基部分对接后，HDMA可与半胱氨酸-113形成硫酯键。
