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Ca²⁺-dependent Binding of Tamoxifen to Calmodulin Isolated from Bovine Brain¹

Center for Cell Biology and Center of Hormonology, Faculty of Medicine, University of Coimbra, 3049 Coimbra Codex, Portugal

The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [³H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca²⁺-dependent manner and that two components are involved in the binding, with apparent dissociation constants (K_d) of about 6 nM and 9 µM. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a B_max value of 120 nmol/mg protein. The stimulatory effect of Ca²⁺ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg²⁺. In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity (n_H = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca²⁺ + Mg²⁺)-ATPase. The results indicate that Tx may alter Ca²⁺-dependent processes by interacting directly with CaM.

¹ This work was supported by grants from the National Institute for Scientific Research of the Portuguese Ministry of Education and from the Calouste Gulbenkian Foundation.

² To whom reprint requests should be addressed, at Center for Cell Biology, Department of Zoology, University of Coimbra, 3049 Coimbra, Portugal.

Received 8/23/89. Revised 1/ 4/90.

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