Kosar E, Teisinger J, Vyskocil F, Vanĕcek J
Institute of Physiology, Academy of Sciences of Czech Republic, Prague.
J Neurochem. 1994 Aug;63(2):662-70.
The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for 125I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N-ethylmaleimide and p-chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited 125I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I-melatonin binding to guanine nucleotides after N-ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-acetylimidazole, and p-nitrobenzenesulfonyl fluoride also inhibited 125I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.
在特定条件下，用蛋白质修饰剂处理膜结合型或可溶型褪黑素受体，然后检测其对¹²⁵I - 褪黑素的结合能力，以获取配体结合域中存在的氨基酸信息。对巯基具有特异性的试剂（N - 乙基马来酰亚胺和对氯汞苯甲酸）、胍基（苯乙二醛）以及氨基（4,4'-二异硫氰酸根合芪 - 2,2'-二磺酸和1 - 氟 - 2,4 - 二硝基苯）以剂量依赖的方式抑制¹²⁵I - 褪黑素的结合，并且它们的作用可通过用冷褪黑素预处理来防止。这些结果表明褪黑素结合域中存在半胱氨酸、精氨酸和赖氨酸残基。N - 乙基马来酰亚胺预处理后，¹²⁵I - 褪黑素与鸟嘌呤核苷酸结合的敏感性降低，这表明受体与G蛋白之间的偶联域内存在另一个巯基。酪氨酸试剂四硝基甲烷、7 - 氯 - 4 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂茂、N - 乙酰咪唑和对硝基苯磺酰氟也抑制¹²⁵I - 褪黑素的结合，并且它们的作用可通过冷褪黑素预处理来防止；然而，它们仅在可能与巯基发生交叉反应的浓度下才有效。组氨酸试剂焦碳酸二乙酯以剂量依赖的方式抑制¹²⁵I - 褪黑素的结合，并且其作用可被冷褪黑素逆转。然而，焦碳酸二乙酯在可溶型受体制剂中的作用较小，因此，它可能修饰了远离配体结合位点的位点。我们的数据并不表明在配体结合域中存在色氨酸、天冬氨酸或谷氨酸残基。
