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. Vin- cristine has been shown to inhibit the self-assembly of tubulin into microtubules at substoichiometric concen- trations. The sedimentation velocity patterns at low vincristine concentration (<I X M to 7 X M) consist of a bimodal boundary with a 5.8 S peak and a fast moving peak, with a nominal sz0+ 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 ( 3 X M), most of the protein is polymerized and sediments as a hypersharp peak with a nominal sz0+ value of -20 S. The association constant for the binding of vincristine to tubulin, deter- mined by spectrofluorometry, is 3.5 X lo4 liters/mol at 25 “C. The binding of vincristine does not induce any significant conformational changes in tubulin; how- ever, the difference spectral results indicate perturba- tion of both vincristine and protein chromophores. tubulin at a rate much faster than that of colchicine binding,while Wilson (1970) has reported that high concentrations of VCR induce nearly complete stabilization of the tubulin-col-chicine binding activity. With the above findings in view, it seemed of interest to investigate the mechanism of binding of VCR to tubulin, the VCR-induced tubulin self-association, and the energetics of these interactions. The results of such a study are the subject of this paper. Preliminary reports of this work have been presented earlier (Prakash and Timasheff, 1980, a and b).