CHARACTERIZATION OF HIGH-AFFINITY MELATONIN BINDING-SITES IN PURIFIEDCELL-NUCLEI OF RAT-LIVER

High-affinity 2-I-125-iodomelatonin binding sites in homogenates of pu rified cell nuclei from rat liver were localized and characterized usi ng biochemical binding techniques. Binding at these sites was found to be rapid, reversible, saturable, and to demonstrate pharmacological s electivity. At 0 degrees C, binding reached equilibrium in about 10 mi n. Scatchard analysis of the data at equilibrium revealed a single cla ss of binding sites with a dissociation constant of K-D = 190 +/- 47 p M, B-max = 9.8 +/- 0.6 fmol/mg protein, and a Hill coefficient of n(H) = 1.02 +/- 0.034. Kinetic analysis of the association and dissociatio n curves indicated a kinetic K-D = 148 +/- 41 pM, which is in good agr eement with the value obtained at equilibrium. The specific binding of 2-I-125-iodomelatonin (45 pM) (0.51 +/- 0.04 fmol/mg protein) was sig nificantly improved (0.79 +/- 0.04 fmol/mg protein) when the homogenat es of purified liver cell nuclei were preincubated with DNase (2 mu g/ ml at 37 degrees C for 20 min) before being used in binding experiment s. After the addition of either proteinase K or trichloroacetic acid t o DNase-treated purified cell nuclear homogenates, the specific bindin g disappeared. This suggests that the specific binding of 2-I-125-iodo melatonin in liver cell nuclei is associated with nuclear protein. Com petition experiments show that N-acetyl-serotonin (K-i = 81.3 nM) was more potent than 5-hydroxytryptamine (K-i > 1 mu M) and 5-methoxytrypt amine (K-i > > 10 mu M) in inhibiting 2-I-125-iodomelatonin binding (K -i melatonin = 146 pM). These data indicate that specific 2-I-125-iodo melatonin binding sites exist in the cell nuclei of rat liver, and tha t they may comprise a locus for the intracellular action of melatonin. The correlation between the K-D and B-max values with melatonin conce ntrations in nuclei suggest that these binding sites may be a physiolo gical melatonin receptor, which could explain the described genomic ef fects of the pineal hormone.