Chang R S, Lotti V J, Chen T B
Biochem Pharmacol. 1987 May 15;36(10):1709-14. doi: 10.1016/0006-2952(87)90057-8.
The binding of 3HL-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. 3HL-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound 3HL-364,718. Specific 3HL-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific 3HL-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific 3HL-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that 3HL-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by 3HL-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.
研究了一种极其强效的非肽类胆囊收缩素（CCK）受体拮抗剂3HL-364,718（3S(-)-N-(2,3-二氢-1-甲基-2-氧代-5-苯基-1H-1,4-苯并二氮杂卓-3-基)-1H-吲哚-2-甲酰胺）与大鼠胰腺中经洋地黄皂苷增溶的CCK受体的结合特性。采用聚乙二醇沉淀法，随后通过快速过滤分离游离和结合的3HL-364,718，来测定3HL-364,718与经洋地黄皂苷增溶的受体的结合情况。3HL-364,718与增溶受体的特异性结合取决于洋地黄皂苷和受体浓度，在最佳条件下，特异性结合占总结合的90%以上。Scatchard分析表明存在一类结合位点，其解离常数（Kd）为0.53 nM，最大结合量（Bmax）为3.1 pmol/mg蛋白质。CCK受体激动剂和拮抗剂均以立体特异性方式抑制3HL-364,718与增溶CCK受体的特异性结合。增溶后，各种拮抗剂取代特异性3HL-364,718结合的亲和力与膜结合受体的情况相似；然而，CCK激动剂的亲和力降低了10至100倍。总体而言，所呈现的数据表明，3HL-364,718是一种新的拮抗剂配体，在测定经洋地黄皂苷增溶的受体时，相对于激动剂配体[125I]CCK具有明显优势。对经洋地黄皂苷增溶的CCK受体进行凝胶过滤，随后进行3HL-364,718结合测定，结果显示估计分子量为400,000道尔顿。
