Abstract
A fluorescent vinblastine derivative, vinblastine-4'-anthranilate, has been shown to inhibit polymerization of rat brain tubulin (IC50 = 4.8 microM). Binding of the drug to tubulin increases fluorescence intensity, causes a small emission blue shift, and has a quantum yield of 0.037. Fluorescence increases as a function of drug concentration, with a high affinity site and an undetermined number of lower affinity sites. Photolabeling, by exciting the fluorescent drug-tubulin complex at the absorption maximum of anthranilate, yields a covalent adduct confined to beta-tubulin. Its formation is specific in that it is blocked by maytansine or vinblastine. Tryptic hydrolysis identifies a single fluorescent beta-peptide coinciding with residues 175-213. The interactions between various ligands at this central portion of beta-tubulin are discussed.
Highlights
Vinblastine can bind to dimeric tubulin, to microtubule ends, and to certain aggregates formed by self-association of tubulin
VLB has major effects on the cross-linking of specific SH groups on -tubulin with the bifunctional N,NЈ-ethylene bis(iodoacetamide): Cys239–Cys354 cross-linking is enhanced by VLB, whereas Cys12–Cys201 or Cys211 is inhibited by VLB [8]
We have re-examined VLB binding to tubulin using a photosensitive derivative that absorbed outside the protein wavelength range and that was as small a substituent as possible because large spacers may lead to erroneous localization in tubulin [12]
Summary
Vol 271, No 25, Issue of June 21, pp. 14707–14711, 1996 Printed in U.S.A. (Received for publication, January 19, 1996, and in revised form, April 9, 1996). Vinblastine can bind to dimeric tubulin, to microtubule ends, and to certain aggregates formed by self-association of tubulin This has led to considerable confusion in the literature regarding the affinity constants, which are stated to vary over several orders of magnitude. Addition of a nitro group to the photoactive label linked to the vindoline moiety of vindesine, an active analogue of VLB, circumvented this problem, but labeling still favored ␣- over -tubulin Because of these uncertainties, we have re-examined VLB binding to tubulin using a photosensitive derivative that absorbed outside the protein wavelength range and that was as small a substituent as possible because large spacers may lead to erroneous localization in tubulin [12]. The fluorescent adduct of VLB so produced has both a satisfactory antimicrotubule activity and a quantum yield sufficient to permit localization of VLB binding to -tubulin
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