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.

Oligoanions such as sodium triphosphate or GTP prevent and͞or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastineinduced tubulin polymerization and abolishes the anion effect. Only the ␤-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the ␣-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 M block cleavage of the ␣-tubulin C terminus in the polymer, whereas cleavage of the ␤-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pHdependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastineinduced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the ␤-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.Tubulin is the main structural component of microtubules and is the target of antitumor drugs such as vinblastine, colchicine, and Taxol, which interfere with microtubule function. Vinblastine affects tubulin differently at different concentrations: at substoichiometric concentrations (Ͻ1 M), it diminishes microtubule dynamics (1); at intermediate concentrations (Ͻ10 M), it inhibits the formation of microtubules (2); and at Ͼ10 M, it promotes polymerization or aggregation into spirals and other polymers (3-5). In previous studies (6), we showed that vinblastine-induced tubulin polymerization proceeds in two steps, the formation of smaller oligomers followed by polymerization to larger structures. Oligoanions such as GTP, sodium triphosphate or suramine inhibit the formation of the bigger polymers (6). The region in the protein where these oligoanions interact is not known. We concluded that vinblastine-induced polymerization was electrostatically regulated.Both the ␣ and ␤ monomers of tubulin are acidic showing a high surface charge density. The C termini of both monomers have the highest charge density and can be cleaved with subtilisin at the extreme C-terminal portions (7,8). Subtilisin cleaves ␣-tubulin between . The cleaved protein is referred to as tubulin S or ␣ s ␤ s , and because of the more rapid cleavage of ␤ tubulin, its formation proceeds through an intermediate called ␣␤ s . Both the ␣-tubulin and ␤-tubulin C termini are rich in negatively charged amino acid residues and these have been shown to play a major role in microtubule assembly on the basis o...

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