Kinetic analysis of cooperativity in tubulin polymerization in the presence of guanosine di- or triphosphate nucleotides.

In vitro polymerization of pig brain tubulin, highly purified and deprived of microtubule-associated proteins, was followed by turbidimetry. Treatment of the data yielded the relation existing between the observed turbidity and the amount of polymer formed. This allowed a kinetic analysis, according to Oosawa's theories, of the polymerization process, which consisted of a slow spontaneous nucleation followed by the growth process. The apparent elongation rate constant was closely related to the nucleation process and exhibited a highly cooperative variation with tubulin concentration. The cooperativity was indicative of the size of the nucleus which appears to remain the same whether sheets or microtubules are formed. Magnesium ions appear to play a role in the polymorphism of tubulin polymers, the proportion of microtubules to sheets increasing with magnesium ion concentration. From kinetic experiments evidence was provided for GDP binding in competition with GTP, with a sixfold lower affinity. The tubulin-GDP complex could participate in microtubules elongation, but was not able to form nuclei. The critical concentration of tubulin in the presence of GDP was roughly twice as high as in the presence of GTP.