Specific binding of 2-[¹²⁵I]melatonin by partially purified membranes of rat thymus

Melatonin binding sites were characterized in partially rat thymus membranes. The specific binding of 2-[¹²⁵I]iodomelatonin ([¹²⁵I]MEL) to thymus membranes was dependent on time and temperature, stable, saturable, and reversible. Concentration-dependent binding of [¹²⁵I]MEL to thymus membranes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class binding to sites. The K_d for this single site was 0.47 nM with a binding capacity of 1.01 pM. In competition studies, the specific binding of [¹²⁵I]MEL to thymus membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that, unlike in a saturation studies with [¹²⁵I]MEL, data were compatible with the existence of two classes of binding sites: a high-affinity site with a K_d of 1.72 ± 0.25 nM and a binding capacity of 1.40 ± 0.18 pM, and a low-affinity site with a K_d of 1226 ± 325 Nm and a binding capacity of 460 ± 87 nM. Interestingly, K_d and BC values of the high-affinity binding site described by competition studies are similar to those obtained by saturation studies with [su125I]MEL. Binding of [¹²⁵I]MEL to thymus membranes was specific as indicated by the fact no other precursor or derivative was as potent as melatonin in inhibiting the binding of [¹²⁵I]MEL to membranes. Results strongly that melatonin is involved in regulation of thymus activity.