Anion-induced increases in the rate of colchicine binding to tubulin

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Abstract
The rate of binding of colchicine to [rat brain] tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs only at low concentrations of colchicine (0.6 .times. 10-5 M). The rate increase for sulfate and L-(+)-tartrate is approximately 2.5-fold at 1.0 mM and plateaus at a limiting value of approximately 4-fold at 100 mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 .times. 106 and 2.1 .times. 107 M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 .times. 107 M-1. The affinity constant calculated from steady-state measurements is 3.2 .times. 106 M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine and 1-anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment were detected by circular dichroism or intrinsic fluorescence. The ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. Anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.

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