Fig. 4.

Comparison of compound dissociation rates from tubulin, measured indirectly by determining subsequent inhibition of binding of [³H]colchicine. Reaction mixtures (5 ml) contained the components described previously that stabilize tubulin [8, 18], 1.0 μM tubulin (0.1 mg/ml), and inhibitory compounds at 10 μM: thiocolchicine (◇), combretastatin A-2 (△), MTPT (●), or PAB (○). The reaction mixtures were incubated at 37 °C for 30 min and chilled on ice. Aliquots (0.1 ml) of each reaction mixture were distributed into tubes, and 200 pmol of [³H]colchicine (10 μ1) were added to each tube. Samples were then incubated for the indicated times in triplicate at 37 °C. At each time point, the amount of [³H]colchicine bound to tubulin was determined, and the reaction mixtures with inhibitor were compared with the control containing only [³H[colchicine, with the data expressed as the percentage of inhibition of colchicine binding.