Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [gamma-32P,3H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-Pi-microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s-1 for the cleavage of the gamma-phosphate and 0.02 s-1 for the liberation of Pi, assuming a simple random model. Apparent rate constants for GTP hydrolysis and Pi release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed.