3'-(P-AZIDOBENZAMIDO)TAXOL PHOTOLABELS THE N-TERMINAL 31 AMINO-ACIDS OF BETA-TUBULIN

Taxol possesses an unusual chemical structure, a unique mechanism of a ction, and demonstrated activity in human malignancies. It is the only antitumor agent that has a binding site on the microtubule polymer. T he interaction of Taxol with the microtubule polymer results in the fo rmation of stable bundles of cellular microtubules that are resistant to depolymerization. Although it has become evident that the microtubu le, specifically beta-tubulin, is the target for Taxol, no information is available on the binding site for the drug. In this report, we dem onstrate that 3'-(p-azidobenzamido)taxol, an analogue with similar bio logical activities as Taxol, covalently binds to the N-terminal domain of beta-tubulin after irradiation of the microtubule-drug complex. Ta xol competes with [H-3]3'-(p-azidobenzamido)taxol binding, suggesting that the photoaffinity analog and Taxol are binding at the same or ove rlapping sites. Formic acid cleavage of [H-3]3'-(p-azidobenzamido)taxo l-photolabeled beta-tubulin and subsequent protein sequence and mass a nalyses have identified the N-terminal 31 amino acids as the major sit e for [H-3]3'-(p-azidobenzamido)taxol photoincorporation.