Photoaffinity labeling of tubulin subunits with a photoactive analog of vinblastine

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Abstract
A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N''-(.beta.-aminoethyl)vindesine ([3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 .mu.M NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other''s binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 .mu.M for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 .mu.M for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the .alpha.- and .beta.-subunits of tubulin with increasing concentrations of [3H]ABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an .alpha.:.beta. ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of .alpha.-tubulin and .beta.-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.