Rivkees S A, Conron R W, Reppert S M
Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Boston 02114.
Endocrinology. 1990 Sep;127(3):1206-14. doi: 10.1210/endo-127-3-1206.
Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.
用去污剂洋地黄皂苷溶解蜥蜴脑组织后，使用125I标记的褪黑素（[125I]MEL）对蜥蜴脑中的褪黑素受体进行了鉴定和表征。对溶解后的物质进行饱和研究，发现了一个高亲和力结合位点，其表观平衡解离常数为181±45 pM。结合是可逆的，褪黑素及其密切相关的类似物可抑制结合，但5-羟色胺或去甲肾上腺素则不能。用不可水解的GTP类似物鸟苷5'-（3-O-硫代三磷酸）（GTP-γ-S）处理溶解后的物质，可显著降低受体亲和力。对溶解的褪黑素受体进行凝胶过滤层析，发现了一个高亲和力、大分子量（400,000）的特异性结合峰。在溶解前用GTP-γ-S预处理，导致洗脱的是一个亲和力较低、分子量较小（150,000）的特异性结合峰。为了纯化溶解的受体，通过将6-羟基褪黑素与环氧活化的琼脂糖6B偶联，开发了一种新型亲和层析树脂。使用这种树脂，褪黑素受体被纯化了约10,000倍。纯化后的物质保留了褪黑素受体的药理特异性。这些结果表明，经去污剂处理后能结合配体的褪黑素受体可以被溶解，并通过亲和层析进行大量纯化。
