Hung S C, Ghali N I, Venton D L, Le Breton G C
Biochim Biophys Acta. 1983 Feb;728(2):171-8. doi: 10.1016/0005-2736(83)90468-6.
In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.
在本研究中，我们对血栓素A2/前列腺素H2拮抗剂反式-13-氮杂前列腺酸（13-APA）与分离的人血小板膜之间的相互作用进行了表征。在这些研究中，我们开发了一种以反式[3H]13-APA作为配体的结合测定法。发现反式[3H]13-APA特异性结合迅速、可逆、可饱和且依赖温度。对结合数据进行Scatchard分析得到一条曲线，表明存在两类结合位点：一类高亲和力结合位点，估计解离常数（Kd）为100 nM；另一类低亲和力结合位点，估计Kd为3.5 μM。在饱和状态下，约1 pmol/mg蛋白质的[3H]13-APA与高亲和力位点结合。为了进一步表征[3H]13-APA结合位点的性质，我们评估了顺式13-APA、顺式15-APA、前列腺素F2α、U46619、6-酮前列腺素F1α和血栓素B2的竞争性结合。发现[3H]13-APA结合位点具有立体特异性和结构特异性。因此，13-APA的顺式异构体对结合的亲和力显著降低。此外，不具有生物活性的前列腺素衍生物血栓素B2和6-酮前列腺素F1α也不竞争[3H]13-APA结合。另一方面，作为血栓素A2/前列腺素H2模拟物的U46619和作为血栓素A2/前列腺素H2拮抗剂的前列腺素F2α都有效地竞争[3H]13-APA结合。这些发现表明反式13-APA与血小板膜上的一个特定位点结合，该位点可能代表血栓素A2/前列腺素H2受体。
