The interaction of vincristine with calf brain tubulin.

Abstract

The interaction of the antimitotic drug vincristine with tubulin has been investigated by the techniques of self-assembly, velocity sedimentation, fluorescence, circular dichroism, and differential spectroscopy. Vincristine has been shown to inhibit the self-assembly of tubulin into microtubules at substoichiometric concentrations. The sedimentation velocity patterns at low vincristine concentration (less than 1 X 10(-5) M to 7 X 10(-5) M) consist of a bimodal boundary with a 5.8 S peak and a fast moving peak, with a nominal S20,w value of 9 S. The data conform to the ligand-promoted self-association theory of Cann and Goad (Cann, J.R., and Goad, W.B. (1972) Arch. Biochem. Biophys. 153, 603-609). At higher vincristine concentrations (greater than 8 X 10(-5 M), most of the protein is polymerized and sediments as a hypersharp peak with a nominal S20,w value of approximately 20 S. The association constant for the binding of vincristine to tubulin, determined by spectrofluorometry, is 3.5 X 10(4) liters/mol at 25 degrees C. The binding of vincristine does not induce any significant conformational changes in tubulin; however, the difference spectral results indicate perturbation of both vincristine and protein chromophores.