Brown L, Erdmann E
Biochem Pharmacol. 1983 Nov 1;32(21):3183-90. doi: 10.1016/0006-2952(83)90202-2.
The specific binding of three cardiac glycosides, 3H-ouabain, 3H-digitoxin and 3H-dihydrodigitoxin, to beef cardiac (Na+ + K+)-ATPase was compared. Non-specific binding was defined as that in the presence of 0.1 mM unlabelled compound, or in the absence of ligands. The dissociation constants (KD-values) calculated from the inhibition of 3H-ouabain binding were: ouabain, 2.9 X 10(-9)M; digitoxin, 1.1 X 10(-9)M; and dihydrodigitoxin 2.7 X 10(-8)M. The concentrations which inhibited beef cardiac (Na+ + K+)-ATPase by 50% were: ouabain, 5.9 X 10(-9)M; digitoxin, 1.6 X 10(-9)M; and dihydrodigitoxin, 2.5 X 10(-8)M. Ouabain and digitoxin showed straight Scatchard plots for one site of high affinity (ouabain, KD = 2.6 X 10(-9)M; digitoxin, KD = 1.7 X 10(-9)M). However, dihydrodigitoxin gave a curved Scatchard plot. Analysis of this binding by the methods of M. J. Weidemann, H. Erdelt and M. Klingenberger (Eur. J. Biochem. 16, 313 (1970) for two binding sites gave the following results: for Mg2+,Pi-supported binding, the KD of the high affinity site was 1.6 X 10(-8)M with a capacity similar to that for ouabain of about 30 pmole/mg protein. For binding supported by Na+,ATP,Mg2+, the KD-value of the high affinity site was 5.3 X 10(-8)M of similar capacity. The low affinity binding site (KD = 4.0 X 10(-6)M for Mg2+,Pi; KD = 5.5 X 10(-6)M for Na+,ATP,Mg2+) bound about 350 pmole/mg protein. The low affinity site but not the high affinity site was also present in heat-denatured enzyme. Binding supported by Mg2+,Pi showed one low affinity site only for ouabain and dihydrodigitoxin in the presence of 200 mM Na+. The high affinity sites for these three cardiac glycosides were further characterized by measurement of the association and dissociation rate constants. The specific binding of 3H-ouabain and 3H-dihydrodigitoxin to human cardiac (Na+ + K+)-ATPase was measured. 3H-Ouabain showed a straight Scatchard plot for one high affinity site only (KD = 4.5 X 10(-9) M, capacity about 15 pmole/mg protein). 3H-Dihydrodigitoxin gave two binding sites: a high affinity site (KD = 1.8 X 10(-8) M) of similar capacity to ouabain, and a low affinity site (KD = 2.0 X 10(-6) M) of about 10-fold greater capacity.(ABSTRACT TRUNCATED AT 400 WORDS)
比较了三种强心苷3H-哇巴因、3H-洋地黄毒苷和3H-双氢洋地黄毒苷与牛心肌（Na⁺+K⁺）-ATP酶的特异性结合。非特异性结合定义为在存在0.1 mM未标记化合物时或不存在配体时的结合。根据对3H-哇巴因结合的抑制作用计算出的解离常数（KD值）为：哇巴因，2.9×10⁻⁹M；洋地黄毒苷，1.1×10⁻⁹M；双氢洋地黄毒苷，2.7×10⁻⁸M。抑制牛心肌（Na⁺+K⁺）-ATP酶活性50%的浓度为：哇巴因，5.9×10⁻⁹M；洋地黄毒苷，1.6×10⁻⁹M；双氢洋地黄毒苷，2.5×10⁻⁸M。哇巴因和洋地黄毒苷对一个高亲和力位点呈现直线型Scatchard图（哇巴因，KD = 2.6×10⁻⁹M；洋地黄毒苷，KD = 1.7×10⁻⁹M）。然而，双氢洋地黄毒苷给出的是曲线型Scatchard图。采用M. J. 魏德曼、H. 埃尔德特和M. 克林根贝格尔的方法（《欧洲生物化学杂志》16, 313 (1970)）对这种结合进行两个结合位点的分析，结果如下：对于Mg²⁺、Pi支持的结合，高亲和力位点的KD为1.6×10⁻⁸M，容量与哇巴因的相似，约为30 pmol/mg蛋白质。对于由Na⁺、ATP、Mg²⁺支持的结合，高亲和力位点的KD值为5.3×10⁻⁸M，容量相似。低亲和力结合位点（对于Mg²⁺、Pi，KD = 4.0×10⁻⁶M；对于Na⁺、ATP、Mg²⁺，KD = 5.5×10⁻⁶M）结合约350 pmol/mg蛋白质。热变性酶中也存在低亲和力位点，但不存在高亲和力位点。在200 mM Na⁺存在下，Mg²⁺、Pi支持的结合对哇巴因和双氢洋地黄毒苷仅显示一个低亲和力位点。通过测量缔合和解离速率常数进一步表征了这三种强心苷的高亲和力位点。测定了3H-哇巴因和3H-双氢洋地黄毒苷与人心脏（Na⁺+K⁺）-ATP酶的特异性结合。3H-哇巴因仅对一个高亲和力位点呈现直线型Scatchard图（KD = 4.5×10⁻⁹M，容量约为15 pmol/mg蛋白质）。3H-双氢洋地黄毒苷给出两个结合位点：一个高亲和力位点（KD = 1.8×10⁻⁸M），容量与哇巴因相似，还有一个低亲和力位点（KD = 2.0×10⁻⁶M），容量约大10倍。（摘要截断于400字）
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