Effect of colchicine binding on the reversible dissociation of the tubulin dimer.

The reversible subunit-dissociation equilibria of the tubulin alpha beta dimer and of the colchicine-tubulin dimer complex have been examined by equilibrium ultracentrifugation at 4.6 degrees C. The dissociation constants (KD) of tubulin from bovine brain and from flagellar outer-doublet microtubules of the sea urchin Strongylocentrotus purpuratus were 7.4 x 10(-7) M and 15.2 x 10(-7) M, respectively. In contrast, both brain and outer-doublet colchicine-tubulin complexes dissociated less readily into their alpha- and beta-tubulin monomers; KD = 2.7 x 10(-7) M for the brain complex and KD = 5.0 x 10(-7) M for the outer-doublet colchicine-tubulin species. Podophyllotoxin (2 x 10(-4) M), tropolone (10(-4) M), or both podophyllotoxin and tropolone (2 x 10(-4) and 5 x 10(-4) M, respectively) had no effect on the dissociation constant of brain tubulin. Under these experimental conditions, the initial colchicine-binding capacities of brain and flagellar tubulins were 0.87 +/- 0.05 and 0.70 +/- 0.07 mol/mol, respectively. The colchicine-binding activities of free tubulins decayed at 4 degrees C according to first-order kinetics with half-times of 37 h for brain tubulin and 26 h for flagellar tubulin. However, colchicine-tubulin complexes (brain or flagellar) showed no decay of binding activity when stored at 4 degrees C for periods up to 4 days. These results strongly support the following conclusions: (1) colchicine binding induces or stabilizes a conformational isomer of tubulin that dissociates into its alpha and beta monomers less readily than free tubulin; (2) the conformational change in tubulin is specific for binding of the intact colchicine molecule and does not occur when ligands specific for the trimethoxyphenyl subdomain (i.e., podophyllotoxin) or tropolone subdomain of the colchicine-binding site bind singly or simultaneously to tubulin; and (3) bound colchicine stabilizes tubulin against loss of colchicine-binding activity at 4 degrees C. This altered conformation of tubulin may be involved in the substoichiometric poisoning of microtubule assembly produced by the addition of colchicine-tubulin complexes to the ends of microtubules.