THE ASSEMBLY OF MICROTUBULE PROTEIN INVITRO - THE KINETIC ROLE IN MICROTUBULE ELONGATION OF OLIGOMERIC FRAGMENTS CONTAINING MICROTUBULE-ASSOCIATED PROTEINS

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are, in general, biphasic with 2 major processes of similar amplitude but separated in rate by 1 order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration and are more stringently limited at pH 6.5 than pH 6.95. Such behavior indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. The rate of this process evidently constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAP from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model was derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles were assigned to MAP-containing fragments in both phases of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles were assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behavior with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behavior and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.