[H-3](AZIDOPHENYL)UREIDO TAXOID PHOTOLABELS PEPTIDE AMINO-ACIDS-281-304 OF ALPHA-TUBULIN

The taxoid binding site on porcine brain tubulin was covalently labele d, in the presence or absence of Taxotere, with the photoaffinity reag ent [H-3]-p-(azidophenyl)ureido taxoid derivative [H-3]-TaxAPU [Combea u, C., Commercon, A., Mioskowski, C., Rousseau, B., Aubert, F., & Goel dner, M. (1994) Biochemistry 33, 6676-6683]. After disulfide reduction and carboxymethylation, the alkylated tubulin samples were treated wi th trypsin and the mixtures of peptides were first fractionated by gel filtration over Sephadex G50. Anion exchange chromatography of the ra dioactive areas showed, for one area, three major radioactive signals which were further analyzed by reversed phase C18 HPLC, leading to wel l-resolved radioactive peaks. Microsequencing of these different peaks gave a complete sequence of a tryptic fragment on alpha-tubulin (alph a-281-304) and two partial peptide sequences of a tryptic fragment on beta-tubulin (beta-217-229) in addition to sequences of mixture of pep tides. The radioactive signals were lost while concentrating the sampl es for microsequencing, preventing the identification of the modified amino acids. These results identify the first peptide on alpha-tubulin which binds to the taxoids and confirm the involvement of both alpha- and beta-tubulin in the taxoid binding site.