Vacas M I, Cardinali D P
Horm Res. 1980;13(2):121-31. doi: 10.1159/000179278.
High-affinity binding of melatonin to crude membrane preparations of bovine pineal gland was examined by a rapid filtration procedure through Whatman GFB paper. Maximal melatonin binding was attained in 60 min at 37 degrees C, in 2 h at 25 degrees C and in 5 h at 0 degree C; at 25 and 37 degrees C it was 36 and 42% of that at 0 degree C. Specific binding was thermolabile and decreased following incubation with trypsin; it was also pH dependent, the maximum being observed at physiological pH. Melatonin binding was inhibited by the addition of monovalent or divalent ions to the incubation buffer. Subcellular fractionation studies indicated that 39 and 50% of binding was located in the pellets at 900 and 27,000 g whereas 11% was detected in the microsomal pellet. Scatchard analysis revealed a single population of binding sites with Kd = (7.0 +/- 1.5) 10(-7) M (mean +/- SEM, n = 4); binding site concentration ranged from 185 to 356 fmol/mg of protein. When various melatonin analogues were tested for their ability to inhibit (3H)-melatonin binding, the following Ki values (10(-7) M), were obtained: N-acetyl-serotonin 120, serotonin 130, 2-methyl indole 154, 5-hydroxytryptophol 218, 5-methoxytryptamine 266, 5-methoxytryptophol 660, tryptamine 1,740, 5-hydroxyindole acetic acid 3,455, 5-methoxyindole acetic acid 12,690, 5-hydroxytryptophan 13,600, and 6-hydroxymelatonin 55,550. Those results suggest that melatonin receptors may be present in the pineal gland.
通过Whatman GFB滤纸快速过滤法检测了褪黑素与牛松果体粗制膜制剂的高亲和力结合。在37℃下60分钟、25℃下2小时和0℃下5小时达到最大褪黑素结合；在25℃和37℃时，其结合量分别为0℃时的36%和42%。特异性结合对热不稳定，用胰蛋白酶孵育后会降低；它也依赖于pH值，在生理pH值时观察到最大值。向孵育缓冲液中添加单价或二价离子可抑制褪黑素结合。亚细胞分级分离研究表明，900g和27,000g离心后的沉淀中分别有39%和50%的结合位点，而在微粒体沉淀中检测到11%。Scatchard分析显示存在单一的结合位点群体，Kd = (7.0 +/- 1.5)×10(-7) M（平均值 +/- 标准误，n = 4）；结合位点浓度范围为185至356 fmol/mg蛋白质。当测试各种褪黑素类似物抑制(3H)-褪黑素结合的能力时，获得了以下Ki值（10(-7) M）：N-乙酰血清素120、血清素130、2-甲基吲哚154、5-羟基色醇218、5-甲氧基色胺266、5-甲氧基色醇660、色胺1,740、5-羟基吲哚乙酸3,455、5-甲氧基吲哚乙酸12,690、5-羟基色氨酸13,600和6-羟基褪黑素55,550。这些结果表明松果体中可能存在褪黑素受体。
