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/1 (3‐weeks old, n = 4), which was in good agreement with the Kd (50.6 pmol/1) 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 > 0.05) in the Kd between mid‐light (42.1 ± 3.9 pmol/1) and mid‐dark (31.6 ± 4.9 pmol/1). 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 > 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 < 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.