Kinetics of Colchicine Binding to Purified &Tubulin Isotypes from Bovine Brain*

Tubulin, the constituent protein of microtubules, is an a@ heterodimer; both a and B exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochernis try 17, 4266-4272). We have studied the kinetics of colchicine binding to purified &tubulin isotypes and find that each of the purified &tubulin isotypes binds colchicine in a monophasic manner. The apparent onrate constants for the binding of colchicine to CY&, aflIII-, and a&-tubulin dimers are respectively 132 f 6, 30 f 2, and 236 f 7 M-’ s-l. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 X lo6, 0.12 X lo6, and 3.31 X lo6 M-’, respectively, for aal~-, and abIv-tubulin dimers. Our results are in agreement with the hypothesis that the 8-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.