TY - JOUR
T1 - Characterization of the taxol binding site on the microtubule
T2 - 2-(m-azidobenzoyl)taxol photolabels a peptide (amino acids 217-231) of β-tubulin
AU - Rao, Srinivasa
AU - Orr, George A.
AU - Chaudhary, Ashok G.
AU - Kingston, David G.I.
AU - Horwitz, Susan Band
PY - 1995/9/1
Y1 - 1995/9/1
N2 - Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3′-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels β-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)-taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled β-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of β-tubulin as the major photolabeled domain.
AB - Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3′-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels β-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)-taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled β-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of β-tubulin as the major photolabeled domain.
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U2 - 10.1074/jbc.270.35.20235
DO - 10.1074/jbc.270.35.20235
M3 - Article
C2 - 7657589
AN - SCOPUS:0028861177
SN - 0021-9258
VL - 270
SP - 20235
EP - 20238
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 35
ER -