INTERACTION OF DOLASTATIN-10 WITH TUBULIN - INDUCTION OF AGGREGATION AND BINDING AND DISSOCIATION REACTIONS

We have prepared [H-3]dolastatin 10 and examined its interactions with tubulin. Binding kinetics appeared to be biphasic, with a rapid initi al reaction that could not be accurately measured, followed by a slowe r second reaction. Bound drug was stable in centrifugal gel filtration , column gel filtration, and high performance liquid chromatography ge l filtration, but the bound drug could be displaced by an active isome r of dolastatin 10. Scatchard analysis of binding data was consistent with two classes of binding sites. However, dolastatin 10 induced an a ggregation reaction upon binding to tubulin, complicating analysis of the data, and incorporation of [H-3]dolastatin 10 into large aggregate s was readily demonstrated. The chromatographic properties of the smal lest radiolabeled species that could be documented were most consisten t with a complex consisting of two molecules of alpha/beta-tubulin dim er and two molecules of [H-3]dolastatin 10. The coexistence of an aggr egation reaction with a binding reaction at a single site probably und erlies the biphasic binding kinetics and the biphasic Scatchard plot. Of peptides that strongly inhibit tubulin polymerization (dolastatin 1 0, dolastatin 10 isomers, segments, and analogs, dolastatin 15, and ph omopsin A), only those previously shown to be strong inhibitors of vin blastine binding and nucleotide exchange also strongly inhibited [3H]d olastatin 10 binding and induced tubulin aggregation (dolastatin 10 it self, two chiral isomers of dolastatin 10, and phomopsin A). The morph ology of dolastatin 10-induced aggregates was compared with that of vi nblastine-induced aggregates under a variety of reaction conditions. W ith both drugs the aggregates had a more organized appearance when mic rotubule-associated proteins were included in the reaction.