Detection of specific protein bands with melatonin-like immunoreactivity in different cell lines and human brain regions

The pineal hormone melatonin regulates various neural and endocrine process es involved in mammalian circadian rhythms, To understand how melatonin med iates these functions, we investigated melatonin-like immunoreactivity (MLI ) in cell extracts and human brain. In Western immunoblots, we detected hig h-molecular-mass protein bands (85-135 kDa) that specifically reacted with the anti-melatonin antibody. The specific protein bands were present in cel l extracts from the human brain and cell lines of different origins. The im munoreactive signal of the 135-kDa protein band was highest in a neuroendoc rine PC12 cell line, which was 10-fold higher than the signal observed in a ny cell extracts used, The commercial antibody employed in the Western blot s was further purified against serum proteins and thyroglobulins. We have p reviously reported that the antibody against melatonin recognizes MLI as de tected by a sensitive RIA, In the present report we have detected the putat ive melatonin-specific binding proteins, which could contribute to the MLI, Our results suggest that melatonin binds with specific proteins in differe nt cellular and brain extracts, the protein(s) being maximally synthesized in PC12 cells. These results may indicate a group of yet unknown proteins s haring a melatonin-like epitope or the presence of melatonin-binding protei n(s) that regulate availability of free melatonin, or both.