© 1989 Oxford University Press
research-article |
Allosteric Binding of Nickel(II) to Calmodulin
Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute FCRF, Frederick, Maryland 21701
Received December 27, 1988; accepted June 8, 1989
The binding of Ni(II) to calmodulin (CAM) in the presence and in the absence of Ca(II) was investigated by equilibrium dialysis in order to test the physicochemistry of direct Ni(II)-CAM interactions that might be responsible for the effects of this metal on CAM observed in vivo. Samples containing 5 µm CAM, 5 mM Tris/HCl buffer (pH 7.4), and NaCl to maintain the ionic strength I = 3600 µm, with or without 200 µm CaCl2, were dialyzed at 37°C against 1–300 µm 63NiCl2. In the presence of Ca(II), the CAM molecule has two binding sites for Ni(II) (K1, = 7.25 × 105m–1; = 3.79 × 103 M–1) with marked coopera-tivity (Hill coefficient = 1.20 ± 0.03 SE). In the absence of Ca(II), a complicated Ni(II)-binding curve is obtained indicating formation of many mutually interacting complex species. Binding of Ni(II) to CAM in the presence of Ca(II) is inhibited slightly by added MnCl2 (50 µM) and very strongly by CuCl2 and ZnCl2 (10 µm). To elucidate the mechanism of this inhibition, binding of Zn(II) (0.5–50 µm 65ZnCl2) to CAM in the presence of Ca(II) (200 µM) was also studied. The maximum molecular ratio of Zn(II) to CAM in the Zn(II)/Ca(II)/CAM complex approached 0.5. Thus, the observed inhibition by Zn(II) of the Ni(II) binding to Ca(II)/CAM does not involve competition for the same binding sites but is rather caused by a conformational arrangement of CAM in its Ca(II)/Zn(II) complex that is different than the Ca(II) complex. This fact, as well as the observed difference in binding of Ni(II) in the presence and absence of Ca(II), stress the importance of conformation of the CAM molecule to Ni(II) binding.