Characterization of high-affinity melatonin binding sites in purified cell nuclei of rat liver

Abstract

High-affinity 2-125I-iodomelatonin binding sites in homogenates of purified cell nuclei from rat liver were localized and characterized using biochemical binding techniques. Binding at these sites was found to be rapid, reversible, saturable, and to demonstrate pharmacological selectivity. At 0 degrees C, binding reached equilibrium in about 10 min. Scatchard analysis of the data at equilibrium revealed a single class of binding sites with a dissociation constant of KD = 190 +/- 47 pM, Bmax = 9.8 +/- 0.6 fmol/mg protein, and a Hill coefficient of nH = 1.02 +/- 0.034. Kinetic analysis of the association and dissociation curves indicated a kinetic KD = 148 +/- 41 pM, which is in good agreement with the value obtained at equilibrium. The specific binding of 2-125I-iodomelatonin (45 pM) (0.51 +/- 0.04 fmol/mg protein) was significantly improved (0.79 +/- 0.04 fmol/mg protein) when the homogenates of purified liver cell nuclei were preincubated with DNase (2 micrograms/ml at 37 degrees C for 20 min) before being used in binding experiments. After the addition of either proteinase K or trichloroacetic acid to DNase-treated purified cell nuclear homogenates, the specific binding disappeared. This suggests that the specific binding of 2-125I-iodomelatonin in liver cell nuclei is associated with nuclear protein. Competition experiments show that N-acetyl-serotonin (Ki = 81.3 nM) was more potent than 5-hydroxytryptamine (Ki > 1 microM) and 5-methoxytryptamine (Ki >> 10 microM) in inhibiting 2-125I-iodomelatonin binding (Ki melatonin = 146 pM). These data indicate that specific 2-125I-iodomelatonin binding sites exist in the cell nuclei of rat liver, and that they may comprise a locus for the intracellular action of melatonin. The correlation between the KD and Bmax values with melatonin concentrations in nuclei suggest that these binding sites may be a physiological melatonin receptor, which could explain the described genomic effects of the pineal hormone.