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Chemical Modifications of Melatonin Receptors in Chicken Brain
Author(s) -
Kosař E.,
Teisinger J.,
Vyskočil F.,
Vaněček J.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.63020662.x
Subject(s) - chemistry , melatonin , tetranitromethane , phenylglyoxal , melatonin receptor , cysteine , binding site , amino acid , receptor , histidine , biochemistry , ligand (biochemistry) , arginine , tyrosine , biology , endocrinology , enzyme
The membrane‐bound or solubilized melatonin receptors were treated with protein‐modifying agents under specific conditions and then assayed for 125 I‐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 125 I‐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 125 I‐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 125 I‐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 125 I‐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.