Mechanism of tubulin assembly: role of rings in the nucleation process and of associated proteins in the stabilization of microtubules.
Several types of experiments were designed to elucidate the mechanism of initiation of tubulin assembly in the presence of microtubule-associated proteins (MAPs): (1) The evolution of the proportion of the double ring species with temperature was examined in the ultracentrifuge, under nonpolymerizing conditions (guanosine diphosphate = 0.5 mM). A net dissociation of rings into dimers occurred when temperature increased. The transition took place above 20 degrees C. (2) The kinetic parameters of the exchange at equilibrium between free tubulin dimers and tubulin in rings were studied at different temperatures, using the technique of isotopic exchange at equilibrium with radioactively labeled tubulin. The values found for the half-time of the exchange reaction varied between or approximately 100 min at 0 degrees C and 10 min at 2 degrees C. (3) The relative participation of unlabeled rings and labeled dimers to microtubules in the time course of assembly was studied at 22 degrees C. The time dependence of microtubule specific radioactivity showed that tubulin incorporated in the initial stages of assembly came predominantly from rings (85-90%). This result indicates that oligomers directly issued from rings are the first intermediates in the assembly process and suggests that at the beginning of polymerization incorporation of isomers or fragments of rings proceeds at a faster rate than their dissociation into dimers. (4) Polymerization experiments in the presence of MAPs with increasing concentrations of tubulin dimers indicated that MAPs are in rapid equilibrium with the microtubules and are distributed along the wall in a more or less loose lattice depending on the relative concentrations of tubulin and MAPs in the solution.
D000818:Animals; D001921:Brain / Q000378:metabolism; D004355:Drug Stability; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D008970:Molecular Weight; D011487:Protein Conformation; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D014404:Tubulin