Thermodynamic linkage between tubulin self-association and the binding of vinblastine.
A sedimentation velocity study has been carried out of the vinblastine-induced self-association of calf brain tubulin in PG (0.01 M NaPi and 10(-4) M GTP, pH 7.0) buffer as a function of vinblastine concentration and temperature. The dependence of the weight-average sedimentation coefficients (S20,W) on total protein concentration can be fitted best by an isodesmic, indefinite self-association mechanism. Apparent association constants, derived by computer fittings of the S20,W data, were analyzed in terms of the Wyman linkage equations. Fitting to a variety of reaction models suggested that the self-association is one ligand molecule mediated; i.e., the binding of one vinblastine molecule is coupled to the formation of each intertubulin bond. The intrinsic association equilibrium constant for dimerization of the vinblastine-liganded tubulin was found to be 1.8 x 10(5) M-1. The self-asociation is characterized by an apparent van't Hoff enthalpy change of +8.0 kcal/mol at 5 x 10(-5) M vinblastine and is driven by a positive entropy change. Apparent binding isotherms of vinblastine to tubulin were calculated based on the association mechanism and parameters derived from the linkage analysis and were found to be consistent with the vinblastine binding results previously reported in our laboratory under identical conditions [Lee, J. C., Harrison, D., & Timasheff, S. N. (1975) J. Biol. Chem. 250, 9276---9282]. Comparison of apparent binding curves calculated with different values of the self-association constants suggested that cooperativity between ligand binding and self-association may account for the disparity of the vinblastine-tubulin binding constants reported in the literature.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D008024:Ligands; D008433:Mathematics; D008954:Models, Biological; D011108:Polymers; D011485:Protein Binding; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*; D014747:Vinblastine / Q000378:metabolism*
D008024:Ligands; D011108:Polymers; D014404:Tubulin; D014747:Vinblastine