Tubulin, the constituent protein of microtubules, is an αβ heterodimer; both α and β 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) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified β-tubulin isotypes and find that each of the purified β-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to αβ(II)-, αβ(III)-, and αβ(IV)-tubulin dimers are respectively 132 ± 5, 30 ± 2, and 236 ± 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (K(α)) for binding colchicine. The affinity constants are 0.24 x 106, 0.12 x 106, and 3.31 x 106 M-1, respectively, for αβ(II)-, αβ(III)-, and αβ(IV)-tubulin dimers. Our results are in agreement with the hypothesis that the β-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.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology