Romero M P, García-Pergañeda A, Guerrero J M, Osuna C
Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville,
FASEB J. 1998 Oct;12(13):1401-8. doi: 10.1096/fasebj.12.13.1401.
Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.
褪黑素被认为是钙调蛋白的生理拮抗剂。在本研究中，我们对非洲爪蟾卵母细胞膜中的褪黑素结合位点进行了表征。非洲爪蟾卵母细胞膜对[125I]褪黑素的结合符合与受体位点结合的所有标准。结合作用依赖于时间、温度和膜浓度，且具有稳定性、可逆性、饱和性和特异性。还对该结合位点进行了药理学表征。化学计量学研究显示存在一个高亲和力结合位点，解离常数（Kd）为1.18 nM。这些数据与动力学研究获得的数据（Kd = 0.12 nM）非常吻合。在竞争研究中，我们观察到一个低亲和力结合位点（Kd = 63.41 μM）。此外，该结合位点被表征为钙调蛋白。因此，结合作用依赖于钙，并被抗钙调蛋白抗体以浓度依赖的方式阻断。钙调蛋白抑制剂氯丙嗪也抑制了示踪剂的结合。从这些结果表明，膜结合钙调蛋白在非洲爪蟾卵母细胞中作为褪黑素结合位点，在那里它可能将细胞活动与褪黑素的节律性循环水平联系起来。这一假设与之前将褪黑素描述为钙调蛋白生理拮抗剂的研究结果相关。
