Flutax 1

Comparison of the binding of anti-tubulin antibody and the fluorescent taxol derivative Flutax-1 to the microtubular system of Tetrahymena.[Pubmed:17048696]

Acta Biol Hung. 2006 Sep;57(3):323-9.

Using confocal microscopic analysis, FITC-labelled anti-alpha-tubulin antibody and the fluorescent taxol derivative Flutax-1 in fixed and living Tetrahymena pyriformis GL, longitudinal microtubules, oral and somatic cilia, deep fibers, and contractile vacuole pores were equally labeled. While the antibody stained transversal microtubules, these were not labeled by Flutax-1. At the same time, oral cilia were more intensely stained by Flutax-1, than by the antibody. There were no differences in the staining of fixed preparations and living cells. The observations suggest (i) the difference between the MAPs of longitudinal and transversal microtubules which allow or inhibit the binding of the indicator molecules, and (ii) the different functions of these two types of microtubules.

Fast kinetics of Taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins.[Pubmed:12496245]

J Biol Chem. 2003 Mar 7;278(10):8407-19.

The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.

Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site.[Pubmed:10818101]

J Biol Chem. 2000 Aug 25;275(34):26265-76.

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.