The Interaction of Vincristine with Calf Brain Tubulin

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).