Shoemaker D G, Lauf P K
J Gen Physiol. 1983 Mar;81(3):401-20. doi: 10.1085/jgp.81.3.401.
The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.
研究了强心苷[3H]哇巴因与重新封闭的人红细胞膜的Na +，K +泵的相互作用。在高细胞外Na +培养基中研究了[3H]哇巴因与高细胞内Na +膜的结合，该条件被确定为产生最大的哇巴因结合率。同时检查每个膜结合的哇巴因分子数量和对Na +，K + -ATP酶的相应抑制作用，结果表明一个[3H]哇巴因分子抑制一个Na +，K + -ATP酶复合物。细胞内镁或镁加无机磷酸盐产生最低的哇巴因结合率。如果腺苷酸激酶抑制剂Ap5A阻止通过残留的腺苷酸激酶活性合成三磷酸腺苷（ATP），则二磷酸腺苷（ADP）对哇巴因结合的支持作用可忽略不计。在用台盼蓝抑制内源性核苷二磷酸激酶并通过加入己糖激酶和葡萄糖耗尽残留的ATP后，单独的尿苷5'-三磷酸（UTP）不支持哇巴因结合。仅作用于Na +，K +泵高亲和力结合位点（Km约为1 microM）的ATP促进了最大的[3H]哇巴因结合率。 5'-腺苷-β-γ-亚氨基磷酸酯（AMP-PNP）不能显著刺激哇巴因结合速率，这表明泵的磷酸化是暴露哇巴因受体所必需的。
