PREDOMINANT LABELING OF BETA-TUBULIN OVER ALPHA-TUBULIN FROM PORCINE BRAIN BY A PHOTOACTIVATABLE TAXOID DERIVATIVE

An [(azidophenyl)ureido] taxoid (TaxAPU) was synthesized in a radiolab eled form by coupling an aminotaxoid to tritiated N-methyl-N-(chlorofo rmyl)-p-azidoaniline TaxAPU was used to photolabel polymerized porcine brain tubulin. This newly synthesized probe possesses taxoid properti es as demonstrated by its effect, in the absence of light, on the kine tics of tubulin assembly and microtubule disassembly and on the critic al concentration of tubulin. TaxAPU apparently competes with Taxol for the same binding site with an equilibrium dissociation constant of 6 mu M. The photoactivation of 266 nm of the radiolabeled probe in the p resence of microtubules led to a covalent incorporation of radioactivi ty. Analysis of the radiolabeled polypeptides by electrophoresis under denaturing conditions revealed a specific incorporation of tritium in both the alpha- and beta-subunits of tubulin. A dependence on probe c oncentration was observed for the irreversible radioactivity incorpora ted into both subunits and maintained essentially a ratio of 2.5:1 bet ween beta/alpha. Therefore, TaxAPU constitutes a true photoaffinity pr obe for the taxoid binding site on microtubules. Our results complemen t those reported by Rao et al. (1992) of photo-cross-linking experimen ts with unmodified Taxol.