Evaluation of antimitotic agents by quantitative comparisons of their effects on the polymerization of purified tubulin

Most antimitotic compounds have highly specific interactions with tubulin, the major protein component of microtubules. It is, therefore, often desirable to characterize interactions of these agents with tubulin. In particular, quantitative comparisons between new and old ("standard") agents, between different classes of agent, and between structural analogs (e.g., for a structure-activity relationship study) are important. Because antimitotic drugs have a variety of effects on tubulin and bind at multiple distinct sites on the protein, the tubulin assembly reaction is probably the only universally applicable reaction that can be analyzed. In my laboratory, we use the assembly of purified tubulin induced by higher concentrations of monosodium glutamate as our basic assay system. This report presents a detailed description of our current routine assay, including the effects of a variety of reaction components on the reaction. In addition, the variety of effects that reaction components can have on the quantitative results obtained with drugs, using the colchicine site drug combretastatin A-4 as a model compound, is described.