Interactions of taxol, microtubule-associated proteins, and guanine nucleotides in tubulin polymerization.

Microtubule-associated proteins (MAPs), guanine nucleotides, and reaction temperature have substantial effects on the interaction of taxol with purified tubulin in 0.1 M glutamate. Without MAPs, taxol induced tubulin to polymerize only if GTP was present and the reaction mixture warmed. GTP was hydrolyzed in tandem with polymerization in stoichiometric amounts, while GDP inhibited both polymerization and hydrolysis. The polymerized material contained few microtubules, consisting mostly of sheets of protofilaments which could be depolymerized at 0 “C. The addition of heat-treated MAPs (with minimal GTPase, ATPase, and nucleoside diphosphate kinase activities) to the reaction had dramatic effects on taxol-induced polymerization. Polymerization now occurred with either GTP or higher temperatures. In the latter case, a single wave of polymerization, little affected by GDP, occurred on warming the reaction mixture. With MAPs and taxol, GTP-dependent polymerization occurred at 0 “C. This was associated with tandem and stoichiometric GTP hydrolysis. Both 0 “C polymerization and hydrolysis were totally inhibited by GDP. Once polymerization had reached a plateau at 0 “C, a second wave of polymerization occurred when the reaction mixture was warmed. This second wave of polymerization was independent of GTP hydrolysis and was little affected by GDP. During this second wave of polymerization, however, there was an apparently unrelated GTPase reaction which could be inhibited by GDP. With MAPs the taxol-induced polymer was largely cold-stable, and the predominant structures formed were microtubules. Morphology was unaffected by the presence of nucleotide or reaction temperature. The requirements for nucleotide, MAPs, or increased reaction temperature could also be satisfied by increasing the glutamate concentration of the reaction.