Affinity labeling of melatonin binding sites in the hamster brain

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was used to identify melatonin binding proteins in synaptosomes from Syrian hamster brain. Incubation of the synaptosomes with BIM resulted in a concentration dependent, irreversible inhibition of 2-¹²⁵I-iodomelationin binding. The radioactive form of BIM, N-Bromoacetyl-2-¹²⁵I-iodo-5-methoxytryptamine (¹²⁵I-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependent manner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of ¹²⁵I-BIM into all three proteins was inhibited by BIM> 2-iodomelatonin> melatonin whereas the melatonin antagonist N-(1,4 dinitrophenyl)-5-methoxytryptamine (ML-23) selectively inhibited the labeling of the 45 kDa protein. These results indicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins.