Ardati A, Gottowik J, Henriot S, Clerc R G, Kilpatrick G J
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
J Neurosci Methods. 1998 Mar 13;80(1):99-105. doi: 10.1016/s0165-0270(97)00210-0.
We present the pharmacological characterisation of the recombinant human corticotropin releasing factor binding protein (hCRF-BP) using a simple assay. In this assay we employed [3H]urocortin as the radioligand and, as a means to separate bound and free ligand, adsorption to activated charcoal. Using this method, approximately 60-70% of total binding was specific. Kinetic analysis revealed that association of specific [3H]urocortin binding was monophasic and slow and that the binding was irreversible. Saturation analysis showed a single saturable site of relatively high density (94 fmol per 10 microl of medium from cells transfected with the recombinant CRF binding protein). The apparent Kd for [3H]urocortin binding of 0.25 nM is similar to previously reported affinities of rat urocortin for hCRF-BP. A range of CRF-related peptides potently competed for specific [3H]urocortin binding. The rank order of potency of these agents was human/rat CRF = urotensin 1 > human urocortin > CRF6-33 > sauvagine > ovine CRF. The non-peptide CRF1 receptor antagonists CP 154,526 (N-butyl-N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]p yri midin-4-yl]-N-ethylamine) and SC 241 ([3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazo lo[4,5-d]pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine) were not active at the highest concentration tested (10(-6) M). We conclude that this is a simple and accurate assay for characterisation of the pharmacology of the recombinant CRF-BP. This assay should assist with further study of the pharmacology and function of the CRF-BP.
我们采用一种简单的检测方法对重组人促肾上腺皮质激素释放因子结合蛋白（hCRF-BP）进行了药理学特性分析。在该检测中，我们使用[3H]尿皮质素作为放射性配体，并采用活性炭吸附法来分离结合态和游离态配体。运用此方法，总结合量的约60 - 70%是特异性的。动力学分析表明，特异性[3H]尿皮质素结合的缔合是单相且缓慢的，并且这种结合是不可逆的。饱和分析显示存在一个相对高密度的单一可饱和位点（每10微升转染了重组CRF结合蛋白的细胞培养基中有94飞摩尔）。[3H]尿皮质素结合的表观解离常数（Kd）为0.25纳摩尔，这与先前报道的大鼠尿皮质素对hCRF-BP的亲和力相似。一系列与促肾上腺皮质激素释放因子（CRF）相关的肽能有效竞争特异性[3H]尿皮质素结合。这些药物的效价顺序为：人/大鼠CRF = 尾加压素1 > 人尿皮质素 > CRF6 - 33 > 蛙皮素 > 绵羊CRF。非肽类CRF1受体拮抗剂CP 154,526（N - 丁基 - N - [2,5 - 二甲基 - 7 - (2,4,6 - 三甲基苯基) - 7H - 吡咯并[2,3 - d]嘧啶 - 4 - 基] - N - 乙胺）和SC 241（[3 - (2 - 溴 - 4 - 异丙基苯基) - 5 - 甲基 - 3H - [1,2,3]三唑并[4,5 - d]嘧啶 - 7 - 基] - 双 - (2 - 甲氧基 - 乙基) - 胺）在最高测试浓度（10^(-6) M）时无活性。我们得出结论，这是一种用于表征重组CRF-BP药理学特性的简单且准确方法。该检测方法应有助于对CRF-BP的药理学和功能进行进一步研究。
