Kinetic evidence for a conformation change of tubulin preceding microtubule assembly.

Abstract

The assembly of microtubule protein has been investigated under conditions where the microtubule seeds concentration was varied. The plot of the apparent first order elongation rate constant with microtubule number concentration strongly deviated from the linearity predicted by Oosawa's condensation model and reached a limit at a high concentration of seeds. The increase in seeds concentration was quantitatively assessed from the histograms of microtubule length distribution and from the parallel increases in the rate of depolymerization at 4 degrees C and steady state rate of GTP hydrolysis at microtubule ends. The limit elongation rate constant was independent of elongating sites and tubulin concentrations and was attributed to the true first order rate constant of a conformation change of tubulin preceding its assembly in microtubules. It is hypothesized that polymerizable tubulin would represent a small fraction only of the tubulin molecules in equilibrium with unpolymerizable species. Microtubule assembly would follow the shift of this equilibrium which is fast as compared to the rate of polymerization at low concentration of elongating sites but becomes rate limiting at higher concentrations.