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

被引:63
作者
COMBEAU, C
COMMERCON, A
MIOSKOWSKI, C
ROUSSEAU, B
AUBERT, F
GOELDNER, M
机构
[1] CEA SACLAY,DEPT BIOL CELLULAIRE & MOLEC,SERV MOLEC MARQUEES,F-91191 GIF SUR YVETTE,FRANCE
[2] UNIV STRASBOURG 1,FAC PHARM,CNRS,URA 1386,CHIM BIOORGAN LAB,F-67401 ILLKIRCH GRAFFENS,FRANCE
关键词
D O I
10.1021/bi00187a038
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mu M. 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.
引用
收藏
页码:6676 / 6683
页数:8
相关论文
共 47 条
[1]   LOW RESOLUTION STRUCTURE OF MICROTUBULES IN SOLUTION - SYNCHROTRON X-RAY-SCATTERING AND ELECTRON-MICROSCOPY OF TAXOL-INDUCED MICROTUBULES ASSEMBLED FROM PURIFIED TUBULIN IN COMPARISON WITH GLYCEROL AND MAP-INDUCED MICROTUBULES [J].
ANDREU, JM ;
BORDAS, J ;
DIAZ, JF ;
DEANCOS, JG ;
GIL, R ;
MEDRANO, FJ ;
NOGALES, E ;
PANTOS, E ;
TOWNSANDREWS, E .
JOURNAL OF MOLECULAR BIOLOGY, 1992, 226 (01) :169-184
[2]  
BAYLEY H, 1984, AZIDES NITRENE REACT, P443
[3]  
BAYLEY H, 1983, LABORATORY TECHNIQUE, V12, P112
[4]   SYNTHESIS OF A PHOTOAFFINITY ANALOG OF TAXOL AS AN APPROACH TO IDENTIFY THE TAXOL BINDING-SITE ON MICROTUBULES [J].
CARBONI, JM ;
FARINA, V ;
RAO, S ;
HAUCK, SI ;
HORWITZ, SB ;
RINGEL, I .
JOURNAL OF MEDICINAL CHEMISTRY, 1993, 36 (04) :513-515
[5]   TAXOL EFFECT ON TUBULIN POLYMERIZATION AND ASSOCIATED GUANOSINE 5'-TRIPHOSPHATE HYDROLYSIS [J].
CARLIER, MF ;
PANTALONI, D .
BIOCHEMISTRY, 1983, 22 (20) :4814-4822
[6]   ASSEMBLY OF PURIFIED GDP TUBULIN INTO MICROTUBULES INDUCED BY TAXOL AND TAXOTERE - REVERSIBILITY, LIGAND STOICHIOMETRY, AND COMPETITION [J].
DIAZ, JF ;
ANDREU, JM .
BIOCHEMISTRY, 1993, 32 (11) :2747-2755
[7]   THERMODYNAMICS OF LIGAND-INDUCED ASSEMBLY OF TUBULIN [J].
DIAZ, JF ;
MENENDEZ, M ;
ANDREU, JM .
BIOCHEMISTRY, 1993, 32 (38) :10067-10077
[8]  
DYE RB, 1993, J BIOL CHEM, V268, P6847
[9]   TURBIDIMETRIC STUDIES OF INVITRO ASSEMBLY AND DISASSEMBLY OF PORCINE NEUROTUBULES [J].
GASKIN, F ;
CANTOR, CR ;
SHELANSKI, ML .
JOURNAL OF MOLECULAR BIOLOGY, 1974, 89 (04) :737-+
[10]   TAXOL PHOTOAFFINITY LABEL - 7-(PARA-AZIDOBENZOYL)TAXOL SYNTHESIS AND BIOLOGICAL EVALUATION [J].
GEORG, GI ;
HARRIMAN, GCB ;
HIMES, RH ;
MEJILLANO, MR .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 1992, 2 (07) :735-738