Binding of vinblastine to stabilized microtubules.
Addition of 2-200 microM vinblastine to microtubules at steady state in vitro causes the microtubules to depolymerize, with the formation of protofilament spirals and other aggregated forms of microtubule protein. The presence of such spirals and protein aggregates, which are difficult to separate from microtubules, has complicated attempts to measure the binding of vinblastine to microtubules. We have found that stabilizing bovine brain microtubules in vitro with dimethyl sulfoxide, taxol, or a combination of dimethyl sulfoxide and taxol prevents or greatly retards the formation of protofilamentous spirals, thus permitting us to measure the binding of vinblastine to intact microtubules. Reciprocal plots of binding data indicate the presence of 1.4-1.7 vinblastine binding sites/mol of tubulin in the microtubule, with a Ka of approximately 3-4 x 10(3) M-1. The Ka value obtained is within 1 order of magnitude of the apparent intrinsic binding constant for the binding of vinblastine to tubulin dimers. The results support the idea that depolymerization of microtubules by intermediate and high concentrations of vinblastine occurs by stoichiometric binding of vinblastine to tubulin along the microtubule surface.
Singer
W D
WD
Department of Biochemistry, University of Kansas, Lawrence 66045.
Jordan
M A
MA
Wilson
L
L
Himes
R H
RH
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D017239:Paclitaxel; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*; D014747:Vinblastine / Q000378:metabolism*
D000470:Alkaloids; D014404:Tubulin; D014747:Vinblastine; D017239:Paclitaxel