Equilibrium and rapid kinetic studies on nocodazole-tubulin interaction.
The interaction between nocodazole and calf brain tubulin in 10(-2) M sodium phosphate, 10(-4) M GTP, and 12% (v/v) dimethyl sulfoxide at pH 7.0 was studied. The number of binding sites for nocodazole was shown to be one per tubulin monomer of 50,000 as a result of equilibrium binding studies by gel filtration and spectroscopic techniques. The presence of microtubule-associated proteins did not significantly affect the binding of nocodazole to tubulin. The apparent equilibrium constant measured at 25 degrees C was (4 +/- 1) X 10(5) M-1. Temperature does not significantly affect the apparent equilibrium constant; hence, the binding of nocodazole to tubulin is apparently entropy driven. Stopped flow spectroscopy was employed to monitor the rate of nocodazole binding under pseudo first order conditions. The effects of temperature and nocodazole concentration were studied. The apparent rate constants were dependent on the concentration of nocodazole in a nonlinear manner. In conjunction with results from structural and thermodynamic studies the kinetic results were interpreted to suggest a mechanism of T + N in equilibrium with TN in equilibrium with T* N, where T and N are tubulin and nocodazole, respectively. T and T* represent two conformational states of tubulin. Furthermore, the kinetic data are consistent with the thermodynamic data only if a model of two parallel similar reactions were considered, one rapid and the other slow. The initial binding step for both the rapid and slow phases was characterized by identical binding constants; however, there was a significant difference in the rates of isomerization. Hence, nocodazole is potentially a useful probe for amplifying differences in solution properties of tubulin subspecies.
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D015739:Nocodazole; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014404:Tubulin / Q000378:metabolism*
D001562:Benzimidazoles; D046911:Macromolecular Substances; D014404:Tubulin; D015739:Nocodazole