Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

^a Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville, Spain

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 K_d of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (K_d=0.12 nM). In competition studies, we observed a low-affinity binding site (K_d=63.41 µM). 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.—Romero, M. P., García-Pergañeda, A., Guerrero, J. M., Osuna, C. Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin. FASEB J. 12, 1401–1408 (1998)

Key Words: membranes pd; CaM • indoleamine • protein kinase

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