[3H](Azidophenyl)ureido Taxoid Photolabels Peptide Amino Acids 281−304 of α-Tubulin

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Abstract
The taxoid binding site on porcine brain tubulin was covalently labeled, in the presence or absence of Taxotere, with the photoaffinity reagent [3H]-p-(azidophenyl)ureido taxoid derivative [3H]TaxAPU [Combeau, C., Commerçon, A., Mioskowski, C., Rousseau, B., Aubert, F., & Goeldner, M. (1994) Biochemistry 33, 6676−6683]. After disulfide reduction and carboxymethylation, the alkylated tubulin samples were treated with trypsin and the mixtures of peptides were first fractionated by gel filtration over Sephadex G50. Anion exchange chromatography of the radioactive areas showed, for one area, three major radioactive signals which were further analyzed by reversed phase C18 HPLC, leading to well-resolved radioactive peaks. Microsequencing of these different peaks gave a complete sequence of a tryptic fragment on α-tubulin (α-281−304) and two partial peptide sequences of a tryptic fragment on β-tubulin (β-217−229) in addition to sequences of mixture of peptides. The radioactive signals were lost while concentrating the samples for microsequencing, preventing the identification of the modified amino acids. These results identify the first peptide on α-tubulin which binds to the taxoids and confirm the involvement of both α- and β-tubulin in the taxoid binding site.