Inhibition of microtubule polymerization by the tubulin–colchicines complex; inhibition of spontaneous assembly

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Abstract
The inhibition of microtubule polymerization by colchicine requires the formation of tubulin-colchicine complexes, and inhibition of polymerization is proportional to the concentration of tubulin-colchicine complexes rather than to the total concentration of cochicine. Because the formation of such complexes is slow relative to polymerization, the kinetics of complex formation obscure the kinetics of inhibition of polymerization. Defined quantities of preformed tubulin-colchicine complexes were taken, relying on their slow dissociation, and these were added to [bovine brain] microtubule protein, which was allowed to polymerize by temperature shift to 37.degree. C. The degree of polymerization was then determined by measurement of turbidity at 400 nm. An appropriate kinetic analysis allowed the effects of inhibitor on initiation and elongation phases of polymerization to be distinguished, without resorting to the use of initiation inhibitors. The results are consistent with a reversible association of tubulin-colchicine complex with microtubule ends blocking further elongation (K1 [inhibition constant] = 0.16 .mu.M). Steady-state measurements suggest that copolymerization of tubulin-cholchicine complex is a minor factor under the conditions used. Little inhibition of initiation was observed, possibly because tubulin-cochicine complex competes with the tubulin dimer, but not with the larger oligomers required for the initiation process.