KINETIC-ANALYSIS OF MICROTUBULE SELF-ASSEMBLY INVITRO

The kinetics of [porcine brain] microtubule assembly were investigated by monitoring changes in turbidity which result from the scattering of incident light by the polymer. Assembly probably occurred by a pathway involving a nucleation phase, followed by an elongation phase as evidenced by a lag in the polymerization kinetics, followed by a psuedo 1st order exponential increase in turbidity. Analytical ultracentrifugation of solutions polymerized to equilibrium showed that 6 S tubulin was the only species detectable in equilibrium with microtubules. The elongation reaction in mixtures of 6 S tubulin and microtubule fragments demonstrated that the net rate of assembly was the sum of the rates of polymerization and depolymerization; the rate of polymerization was proportional to the product of the microtubule number concentration and the 6 S tubulin concentration; and the rate of depolymerization was proportional to the number concentration of microtubules. Microtubule assembly probably occurs by a condensation polymerization mechanism consisting of distinct nucleation and elongation steps. Microtubules are initiated in a series of protein association reactions in a pathway that was not fully elucidated. Elongation proceeds by the consecutive association of 6 S tubulin subunits onto the ends of existing microtubules. Depolymerization occurs by dissociation of 6 S subunits from the ends of microtubules. The rate constants measured for polymerization and depolymerization at 30.degree. C were 4 .times. 106 M-1 s-1 and 7 s-1, respectively.