Abstract
We reported previously that calmodulin binds to tubulin in a Ca2+-dependent manner, thereby inhibiting microtubule assembly. In this work, we quantitatively investigated the binding between calmodulin and tubulin by applying two analytical methods. One was the frontal analysis using affinity chromatography originally developed by Kasai and Ishii (J. Biochem. 84, 1061-1069, 1978). The use of tubulin-Sepharose columns gave a dissociation constant of 4.0 μM. The other was the equilibrium gel filtration developed by Hummel and Dreyer (Biochim. Biophys. Acta 63, 532-534, 1962). This method using a Sephadex G-100 column provided a dissociation constant of 3.5 μM under the same medium conditions as in the frontal analysis, and it was found that 2 mol calmodulin could bind to 1 mol tubulin. Furthermore, the frontal analysis method was convenient for studies on the effect of temperature and ionic strength on the binding. Upon elevating the temperature, the dissociation constant increased. Increase in the ionic strength also increased the dissociation constant.
