CHARACTERIZATION OF THE CHICKEN BRAIN MELATONIN-BINDING PROTEIN USINGIODINATED AND TRITIATED LIGANDS

The melatonin-binding protein in chicken brain membranes was character ized using both [I-125]-2-iodomelatonin and [H-3]-melatonin as radioli gands, Saturation studies conducted at 25 degrees C revealed a single class of binding site with dissociation constants of 24 +/- 4.8 pM (n = 7) and 125 +/- 21 pM (n = 6) for the iodinated and tritiated ligands , respectively. Calculation of the affinity constant using data from k inetic experiments gave values of 2.2 +/- 0.4 pM and 135 +/- 15 pM for the iodinated and tritiated ligands, respectively. Competition studie s showed that the rank order of inhibition of binding by melatonin ana logues was similar for both radioligands (2-iodomelatonin > melatonin > 2,3-dihydromelatonin > N-acetyl-5-methoxykynurenamine > N-acetylsero tonin > 5-methoxytryptamine). The calculation of Ki, which depends upo n the affinity constant, was 22 +/- 4.9 pM and 129 +/- 21 pM for 2-iod omelatonin and melatonin, respectively, when the affinity constant der ived from the [I-125]-2-iodomelatonin saturation experiments was used, but 4.9 +/- 1.5 pM and 33 +/- 5.5 pM when the kinetically derived con stant was used. When [H-3]-melatonin was used, the Ki for melatonin wa s 72 +/- 8 pM and 20 +/- 4.6 pM for 2-iodomelatonin and melatonin. Bin ding of [I-125]-2-iodomelatonin to the membranes was partially reversi ble at 25 degrees C in contrast to the complete reversibility of [H-3] -melatonin. Examination of the effects of temperature on binding indic ated that at 37 degrees C both association and dissociation of both li gands were accelerated. Closer examination showed that at 37 degrees C there was a loss of approximately 40% of the [I-125]-2-iodomelatonin binding sites and little influence upon the affinity of binding with t ime. By contrast, when [H-3]-melatonin was used, the affinity decrease d fourfold, with only a slight change in the number of sites. If membr anes were incubated at 37 degrees C and then switched 25 degrees C, bi nding increased, emphasizing the fact that the binding sites were not destroyed. Whereas there appears to be little doubt that 2-iodomelaton in is a biologically active melatonin agonist, the binding of the radi oactive form of this agonist to the putative melatonin receptor bindin g site is quite different from that of the endogenous ligand. This may have serious consequences in studies where receptor content is determ ined following physiological or pharmacological interventions.