Interaction of vinblastine with steady-state microtubules in vitro

Abstract

At low concentrations, vinblastine binds rapidly and reversibly to a very limited number of high affinity sites on steady-state bovine brain microtubules (mean K d, 1.9 × 10 −6 m; 16.8 ± 4.3 vinblastine binding sites per microtubule) which appear to be located at one or both ends of the microtubules. At high concentrations, vinblastine binds to a high binding capacity class of sites of undetermined affinity, located on helical strands of protofilaments which form at the ends of depolymerizing microtubules, and/or along the surface of the microtubules. Substoichiometric inhibition of microtubule assembly, which occurs at low vinblastine concentrations, appears to be due to the binding of vinblastine to the high affinity class of sites. Fifty per cent inhibition of tubulin addition to the net assembly ends of steady-state microtubules occurred at 1.38 × 10 −7 m-drug, and at this concentration, 1.16 ± 0.27 molecules of vinblastine were bound to the high affinity class of sites. Vinblastine appeared to bind directly to the microtubule ends, and our results indicate that vinblastine inhibits the assembly of steady-state bovine brain microtubules by binding rapidly and with high affinity to one or two molecules of tubulin at the net assembly ends. Splaying and peeling of protofilaments at microtubule ends and the active depolymerization of microtubules occurred only at vinblastine concentrations greater than 1 × 10 −6 to 2 × 10 −6 m. This action of vinblastine is associated with and may be due to the binding of vinblastine to the high capacity class of sites. Both actions of vinblastine may be due to the binding of vinblastine to the same binding sites on the tubulin molecule, with the sites exhibiting either a high or low affinity depending upon the location in the microtubule.