Kinetics of colchicine binding to purified β-tubulin isotypes from bovine brain

A. Banerjee, R. F. Luduena

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Abstract

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 languageEnglish (US)
Pages (from-to)13335-13339
Number of pages5
JournalJournal of Biological Chemistry
Volume267
Issue number19
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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