Calcium binding to the tubulin-colchicine complex in the state of GTP in a BES buffer

Magnesium and calcium binding were assayed using the tubulin-colchicine complex in a BES buffer, in which the calcium binding to tubulin had been measured by Grisham et al. (Fed. Proc. 39, 2162). In the previous paper, an imidazole buffer was used as the buffer which does not chelate to calcium and which is substituted to phosphate buffer. The result of calcium binding measurement indicated the same binding constant between at pH 7.0 and at pH 6.5 in the absence of magnesium (1.08×10^ M at pH 7.0 and 1.10×10^ M at pH 6.5). Also, the calcium binding constant of the tubulin-colchicine complex was the same as that of tubulin in a BES buffer, pH 7.0. The increase of magnesium concentration inhibited calcium binding to the tubulin-colchicine complex. The affinity of calcium at pH 7.0 was lower than that at pH 6.5 in the presence of magnesium. This leads the effect of calcium on the polymerization of the tubulin-colchicine complex is small at pH 6.5 rather than at pH 7.0. The results obtained in the polymerization experiment were in good agreement with the results of the calcium binding experiment