Interaction of nocodazole with tubulin isotypes

Nocodazole is an anti-mitotic drug that has long been used as an experimental tool in cell biology. Although nocodazole is known to bind with high affinity to tubulin and to inhibit microtubule assembly, very little has been done on its precise mechanism of action. In fact, its binding to the different isotypes of tubulin has never been addressed. Although the nocodazole binding site overlaps with that of colchicine, the two drugs are structurally quite different. The tubulin molecule is an alpha/beta heterodimer; both a and P exist as various isotypes whose distribution and drug-binding properties are significantly different. In this study, we measured the binding affinity of nocodazole for purified tubulin isotypes. Using fluorescence quenching analysis, we found that the binding kinetics of nocodazole with each type of tubulin best fits a two-affinity Michaelis-Menten binding model. The apparent dissociation constants for the high-affinity binding sites are 0.52 +/- 0.02 for alphabeta(II), 1.54 +/- 0.29 for alphabeta(III), and 0.29 +/- 0.04 for alphabeta(IV). Thus, nocodazole has the highest affinity for alphabeta(IV) and the lowest affinity for alphabeta(III). Knowledge of the isotype specificity of nocodazole may allow for development of novel therapeutic agents based on this drug. Drug Dev. Res. 55:91-96, 2002. (C) 2002 Wiley-Liss, Inc.