Mechanism for nucleotide incorporation into steady-state microtubules.

We have extended our previous theoretical analysis of the kinetics for radioactive GTP incorporation into steady-state microtubules [Zeeberg, B., Reid, R., & Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899] to include the effects of a kinetic barrier for equilibration of labeled GTP with the tubulin E site. This binding has been found to be relatively slow; the half-time for GTP dissociation is approximately 25 s (k = 0.028 s-1). The slow binding of radioactive GTP apparently accounts for the following observations: (a) more radioactive nucleotide is incorporated into steady-state microtubules in the first 20 s when tubulin-[3H]GTP is used in a pulse than when [3H]GTP is used; (b) when steady-state microtubules are pulsed for 20 s with tubulin-[3H]GTP and then chased with excess nonradioactive GTP, radioactive nucleotide incorporation is not stopped immediately. Quantitative analysis of these results indicates that our steady-state microtubules do not contain significant amounts (greater than 1%) of GDP or GTP which can exchange with added GTP. The principal route for labeled nucleotide incorporation appears to be from tubulin-[3H]GTP subunit uptake, by diffusional and treadmilling processes.