Lowenstein P R, Cardinali D P
Neuroendocrinology. 1983 Aug;37(2):150-4. doi: 10.1159/000123533.
High affinity binding of 3H-flunitrazepam (FNZP) to crude membrane preparations of bovine pineal membranes was examined by a rapid filtration procedure through Whatman GFB paper. At 0 degrees C binding reached equilibrium in about 20 min. Scatchard analysis of data at equilibrium revealed a single population of binding sites with dissociation constant (Kd) = 3.14 +/- 0.45 nM and binding site concentration (Bmax) = 55.6 +/- 5.6 fmol/mg protein. Kinetic analysis of the association and dissociation curves indicated a kinetic Kd = 1.13 nM, in reasonable agreement to that obtained at equilibrium. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit 3H-FNZP binding, the following Ki (nM) were obtained: clonazepam (0.22), Ro 15-1788 (0.48), FNZP (0.95), Ro 5-4864 (greater than 10,000). When the beta-carboline derivative 3H-ethyl-beta-carboline-3-carboxylate ester (E beta CEE) was used as a radioligand, Kd at equilibrium (0.98 nM), kinetic Kd (1.69 nM) and affinity order for analogues were in close agreement to those found for FNZP binding; however, Bmax was about 60% that observed for 3H-FNZP binding. Addition of GABA or pentobarbital (100 microM) to pineal membranes increased 3H-FNZP binding by 55 and 72%. These results suggest the existence of a mixed population of type 1 and type 2 central BZP receptor subclass in bovine pineal gland.
通过Whatman GFB纸快速过滤程序检测了3H-氟硝西泮（FNZP）与牛松果体粗制膜制剂的高亲和力结合。在0℃时，结合在约20分钟内达到平衡。对平衡数据的Scatchard分析显示存在单一的结合位点群体，解离常数（Kd）= 3.14±0.45 nM，结合位点浓度（Bmax）= 55.6±5.6 fmol/mg蛋白质。结合和解离曲线的动力学分析表明动力学Kd = 1.13 nM，与平衡时获得的值合理一致。当测试各种苯二氮䓬（BZP）类似物抑制3H-FNZP结合的能力时，获得了以下Ki（nM）：氯硝西泮（0.22）、Ro 15-1788（0.48）、FNZP（0.95）、Ro 5-4864（大于10,000）。当使用β-咔啉衍生物3H-乙基-β-咔啉-3-羧酸酯（EβCEE）作为放射性配体时，平衡时的Kd（0.98 nM）、动力学Kd（1.69 nM）以及类似物的亲和力顺序与FNZP结合时发现的结果密切一致；然而，Bmax约为3H-FNZP结合时观察到的值的60%。向松果体膜中添加GABA或戊巴比妥（100μM）可使3H-FNZP结合增加55%和72%。这些结果表明牛松果体中存在1型和2型中枢BZP受体亚类的混合群体。
