Effect of ATP on the kinetics of microtubule assembly.
We investigated the role of ATP in the assembly of microtubules. Tubulin, prepared by chromatography on DEAE-cellulose, was nearly devoid of nucleoside diphosphokinase activity. ATP induced assembly in such preparations for a single assembly/disassembly cycle; then further assembly could not be induced by ATP unless the system was supplemented with additional GTP. This suggests that the E-site must contain GTP for polymerization and ATP interacts at a different site on tubulin. Although tubulin can be assembled into microtubules in 1.0 mM GTP, the inclusion of 0.2 mM ATP along with the GTP increases the rate and extent of assembly. The enhancement increased with increasing ATP concentrations. The inclusion of 0.2 mM ATP reduced the critical concentration for tubulin assembly from 1.5 to 0.9 mg/ml. Analysis of assembly rate versus protein concentration suggested that ATP also affects nucleation. Aggregates of tubulin rings formed by warming tubulin in the presence of 1.0 mM ATP and 5.0 mM Mg2+ were capable of initiating assembly in a solution of tubulin which was not able to polymerize. Furthermore, the extent of microtubule formation was dependent on the concentration of aggregated rings added to the solution. We propose that ATP interacts with tubulin at a binding site that is distinct from the N- and E-sites that bind GTP. A function of ATP binding is to stimulate the formation of tubulin rings as nucleation centers for polymerization.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate