Interaction of dolastatin 10 with tubulin: Induction of aggregation and binding and dissociation reactions

R. Bai, G. F. Taylor, J. M. Schmidt, M. D. Williams, J. A. Kepler, George Pettit, E. Hamel

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Abstract

We have prepared [3H]dolastatin 10 and examined its interactions with tubulin. Binding kinetics appeared to be biphasic, with a rapid initial reaction that could not be accurately measured, followed by a slower second reaction. Bound drug was stable in centrifugal gel filtration, column gel filtration, and high performance liquid chromatography gel filtration, but the bound drug could be displaced by an active isomer of dolastatin 10. Scatchard analysis of binding data was consistent with two classes of binding sites. However, dolastatin 10 induced an aggregation reaction upon binding to tubulin, complicating analysis of the data, and incorporation of [3H]dolastatin 10 into large aggregates was readily demonstrated. The chromatographic properties of the smallest radiolabeled species that could be documented were most consistent with a complex consisting of two molecules of α/β-tubulin dimer and two molecules of [3H]dolastatin 10. The coexistence of an aggregation reaction with a binding reaction at a single site probably underlies the bipbasic binding kinetics and the bipbasic Scatchard plot. Of peptides that strongly inhibit tubulin polymerization (dolastatin 10, dolastatin 10 isomers, segments, and analogs, dolastatin 15, and phomopsin A), only those previously shown to be strong inhibitors of vinblastine binding and nucleotide exchange also strongly inhibited [3H]dolastatin 10 binding and induced tubulin aggregation (dolastatin 10 itself, two chiral isomers of dolastatin 10, and phomopsin A). The morphology of dolastatin 10- induced aggregates was compared with that of vinblastine-induced aggregates under a variety of reaction conditions. With both drugs the aggregates had a more organized appearance when microtubule-associated proteins were included in the reaction.

Original languageEnglish (US)
Pages (from-to)965-976
Number of pages12
JournalMolecular Pharmacology
Volume47
Issue number5
Publication statusPublished - 1995
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology

Cite this

Interaction of dolastatin 10 with tubulin : Induction of aggregation and binding and dissociation reactions. / Bai, R.; Taylor, G. F.; Schmidt, J. M.; Williams, M. D.; Kepler, J. A.; Pettit, George; Hamel, E.

In: Molecular Pharmacology, Vol. 47, No. 5, 1995, p. 965-976.

Research output: Contribution to journalArticle