Theis J G, Dellweg H, Perzborn E, Gross R
Department of Pharmacology, Bayer AG, Wuppertal, Germany.
Eur J Pharmacol. 1992 Jun 5;226(2):149-56. doi: 10.1016/0922-4106(92)90176-v.
[3H]BAY U 3405 was used to characterize the effect of acidic and alkaline pH values on the binding of the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor of human platelet membranes. The specific binding of [3H]BAY U 3405 largely increased upon acidification up to pH 5.8. Saturation binding studies revealed an increase in binding affinity without change in the number of binding sites. At pH 7.4 the Kd was 8.7 +/- 3.7 nM (Bmax = 6.6 +/- 0.6 pmol/mg protein) compared to 1.2 +/- 0.2 nM (Bmax = 6.1 +/- 0.6 pmol/mg protein) at pH 5.8. A more than 10-fold higher rate of association was observed at pH 5.8 compared to pH 7.4, while the rate of dissociation showed only minor changes. The kinetically derived dissociation constant was 1 nM (pH 5.8) and 9.6 nM (pH 7.4). The pH dependency of the binding of structurally different non-labelled ligands to the TXA2/PGH2 receptor was evaluated by inhibition studies at pH 5.8 and pH 7.4. BAY U 3405, daltroban, CTA2, and U 46619 showed significantly higher affinities at pH 5.8. In contrast, I-PTA-OH and GR 32191 had a higher affinity at pH 7.4. No significant difference was seen with SQ 29548 at the observed pH values. A second protonable group within the molecules I-PTA-OH, GR 32191, and SQ 29548 might be responsible for the observed differences.
用[3H]BAY U 3405来研究酸性和碱性pH值对人血小板膜血栓素A2/前列腺素H2（TXA2/PGH2）受体结合的影响。在酸化至pH 5.8时，[3H]BAY U 3405的特异性结合大幅增加。饱和结合研究显示结合亲和力增加，而结合位点数量不变。在pH 7.4时，Kd为8.7±3.7 nM（Bmax = 6.6±0.6 pmol/mg蛋白质），相比之下，在pH 5.8时为1.2±0.2 nM（Bmax = 6.1±0.6 pmol/mg蛋白质）。与pH 7.4相比，在pH 5.8时观察到的缔合速率高出10倍以上，而解离速率仅显示出微小变化。动力学推导的解离常数在pH 5.8时为1 nM，在pH 7.4时为9.6 nM。通过在pH 5.8和pH 7.4下的抑制研究，评估了结构不同的未标记配体与TXA2/PGH2受体结合的pH依赖性。BAY U 3405、达曲班、CTA2和U 46619在pH 5.8时显示出显著更高的亲和力。相比之下，I-PTA-OH和GR 32191在pH 7.4时具有更高的亲和力。在观察到的pH值下，SQ 29548没有显著差异。I-PTA-OH、GR 32191和SQ 29548分子中的第二个可质子化基团可能是观察到差异的原因。
