Predominant Labeling of .beta.- over .alpha.-Tubulin from Porcine Brain by a Photoactivatable Taxoid Derivative

Abstract

An [(azidophenyl)ureido]taxoid (TaxAPU) was synthesized in a radiolabeled form by coupling an aminotaxoid to tritiated N-methyl-N-(chloroformyl)-p-azidoaniline. TaxAPU was used to photolabel polymerized porcine brain tubulin. This newly synthesized probe possesses taxoid properties as demonstrated by its effect, in the absence of light, on the kinetics of tubulin assembly and microtubule disassembly and on the critical concentration of tubulin. TaxAPU apparently competes with Taxol for the same binding site with an equilibrium dissociation constant of 6 microM. The photoactivation of 266 nm of the radiolabeled probe in the presence of microtubules led to a covalent incorporation of radioactivity. 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 concentration was observed for the irreversible radioactivity incorporated into both subunits and maintained essentially a ratio of 2.5:1 between beta/alpha. Therefore, TaxAPU constitutes a true photoaffinity probe for the taxoid binding site on microtubules. Our results complement those reported by Rao et al. (1992) of photo-cross-linking experiments with unmodified Taxol.

Tau expression in model adenocarcinomas correlates with docetaxel sensitivity in tumour-bearing mice

“…haxe the propertx of increasinc microtubule stabilitx to lexels incompatible with normal cell metabolism. P-Tubulins are thought to be the major targets of these drugs (Combeau et al 1994) and. consequently.…”

Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin

“…2). Photoaffinity crosslinking studies have localized Taxol binding to the ␤-subunit of tubulin (3)(4)(5)(6)(7) and, specifically, to peptides 1-31 (5), 217-231 (6), and 277-293 (7). Structure-activity relationship studies have defined the contribution of various substituents in the Taxol molecule (reviewed in ref.…”

Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel

“…Docetaxel, as well as the other taxanes, acts by stabilizing microtubules to levels incompatible with normal metabolism. β-tubulins are the targets for the drugs (Combeau et al, 1994) and previous works have indicated that several β-tubulin isoforms show an overexpression in cell lines resistant to taxol (Haber et al, 1995;Jaffrézou et al, 1995).…”