Interaction of a fluorescent derivative of paclitaxel (Taxol) with microtubules and tubulin-colchicine.
A fluorescent derivative of paclitaxel, 2-debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT), has been prepared. 2-AB-PT induces microtubule assembly in vitro, but is about 3-fold less potent than paclitaxel itself. The absorption and emission characteristics of 2-AB-PT were analyzed as a function of solvent. It was found that both spectra were perturbed by specific solvent effects when the solvent contained a hydrogen bond donor. The absorption and fluorescence spectra of 2-AB-PT bound to microtubules could not be mimicked by a single solvent, but the absorption and emission maxima of the tubulin-bound species could be duplicated by a solvent mixture of DMSO and water. These results indicate that the fluorophore binding site on the microtubule is in an environment of intermediate polarity that is accessible to a hydrogen bond donor in the vicinity of the m-amino group. In addition, tubulin fluorescence is quenched in the 2-AB-PT/microtubule complex, and energy transfer from tubulin to 2-AB-PT is apparent. These results indicate that substituents on the C-2 position of paclitaxel associate with tubulin when bound to the microtubule. 2-AB-PT binding to microtubules was quantitatively analyzed by fluorescence titrations. Two classes of binding sites for 2-AB-PT on microtubules were found. The high affinity site has an apparent association constant (K1app) of 2.0 (+/- 0.9) x 10(7) M-1 and an apparent binding stoichiometry (n1app) of 0.8 (+/- 0.1) sites/tubulin dimer in the microtubule. The apparent association constant for the lower affinity site is about 100-fold less than that of the higher affinity site (K2app = 2.1 (+/- 0.7) x 10(5) M-1), and the stoichiometry of the lower affinity site or class of sites (n2app) was found to be 1.3 +/- 0.1. Paclitaxel blocked 2-AB-PT binding to the high affinity site. No binding of 2-AB-PT to unassembled tubulin was observed, but the emission spectrum of 2-AB-PT in the presence of the tubulin-colchicine complex resembled the emission spectrum of the ligand bound to microtubules. It was previously shown that paclitaxel can induce GTPase activity in the tubulin-colchicine complex, indicating that paclitaxel can bind to unassembled tubulin in its complex with colchicine [Carlier, M.-F., & Pantaloni, D. (1983) Biochemistry 22, 4814-4822]. Rigorous characterization of the aggregation state of the protein under these conditions demonstrates that 2-AB-PT is also capable of binding to the tubulin-colchicine complex.
Department of Chemistry, State University of New York at Binghamton 13902-6016, USA.
Y
Y
Han
A G
AG
Chaudhary
M D
MD
Chordia
D L
DL
Sackett
B
B
Perez-Ramirez
D G
DG
Kingston
S
S
Bane
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002474:Cell-Free System; D003078:Colchicine / Q000737:chemistry*; D005456:Fluorescent Dyes; D006860:Hydrogen Bonding; D008870:Microtubules / Q000737:chemistry*; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000737:chemistry*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D043823:Taxoids; D014404:Tubulin / Q000737:chemistry*; D014461:Ultracentrifugation
D005456:Fluorescent Dyes; C095849:N-debenzoyl-N-(3-(dimethylamino)benzoyl)paclitaxel; D012997:Solvents; D043823:Taxoids; D014404:Tubulin; D017239:Paclitaxel; D003078:Colchicine