High affinity specific binding of 2-[125I]iodomelatonin by spleen membrane preparations of chicken.

The binding sites for 2-[125I]iodomelatonin in chicken spleens were characterized. The binding was rapid, stable, saturable, reversible, and of high affinity. Both melatonin and 6-chloromelatonin strongly inhibited the binding. The dissociation constant (Kd) obtained from the Scatchard analysis was 31.4 +/- 5.19 pmol/l (3-weeks old, n = 4), which was in good agreement with the Kd (50.6 pmol/l) calculated from the kinetic study. The maximum number of binding sites (Bmax) was 1.09 +/- 0.11 fmol/mg protein (3-weeks old, n = 4). Twelve 11-week-old chicks were killed in two groups at mid-light or mid-dark. Saturation studies indicated no significant difference (P greater than 0.05) in the Kd between mid-light (42.1 +/- 3.9 pmol/l) and mid-dark (31.6 +/- 4.9 pmol/l). The maximum number of binding sites (Bmax) at mid-light and mid-dark were 1.52 +/- 0.16 and 1.35 +/- 0.08 fmol/mg protein, respectively, with no significant variation (P greater than 0.05) recorded. However, when the whole spleen was taken into consideration, the Bmax per spleen protein of the mid-light samples (253 +/- 36 fmol/spleen protein) was significantly greater than that (129 +/- 16 fmol/spleen protein) of the mid-dark samples (P less than 0.05). This indicated that in our study a diurnal rhythm of the total number of 2-[125I]iodomelatonin binding sites might exist in the chicken spleen.