Assay for Melatonin Receptor-Binding Affinity and Expression in Mitochondria.

The affinity (K_i) of melatonin for melatonin receptors located in B6CBA wild-type mouse brain mitochondria (0.06–0.09 mg protein per tube) and PM (0.24–0.26 mg protein per tube) was assessed by competition binding analysis using 100 pM 2-[¹²⁵I]-iodomelatonin and 1 pM–100 nM melatonin as described (46). Total melatonin receptor expression (B_max) and affinity (K_d) of 2-[^125I]-iodomelatonin binding to melatonin receptors were assessed in mitochondria (0.06–0.22 mg protein per tube) and whole-brain homogenates (0.25–0.46 mg protein per tube) by saturation isotherms using 0–500 pM 2-[^125I]-iodomelatonin for PM and 0–2.4 nM for mitochondria. Nonspecific binding was determined using 10 μM melatonin. Parallel binding assays were performed in the presence of 1 mM DTT using 250–500 pM 2-[I^I25]-iodomelatonin. Data were best-fit analyzed by nonlinear regression analysis least-squares fit (GraphPad Prism, Inc.) (SI Appendix, Fig. S4).