Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.
Biochemistry
; 35(6): 2008-15, 1996 Feb 13.
Article
em En
| MEDLINE
| ID: mdl-8639685
ABSTRACT
The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pirazinas
/
Tubulina (Proteína)
Tipo de estudo:
Risk_factors_studies
Limite:
Animals
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
França