Mechanism for nucleotide incorporation into steady-state microtubules (original) (raw)

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, 9891-98991 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 SI). 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-['HIGTP is used T e incorporation of radioactive guanine nucleotide into microtubules which are at steady state is believed to result from the treadmilling and diffusional uptake of t~bulin-[~H]GTP subunits. The treadmilling component of this process reflects the fact that as a result of GTP hydrolysis, the critical concentrations may be different at the two ends of a microtubule; this allows an excess of tubulin subunits to be added at one end, while an equivalent excess of subunits is lost at the opposite end. Diffusional subunit uptake results from the fact that while one fraction of the microtubule population is undergoing an excess of subunit additions, a different fraction will be undergoing an excess of subunit losses. Although it has been demonstrated that tubulin-bound GDP in the microtubule does not exchange with guanine nucleotide in solution Kobayashi, 1975;, recent results from pulse-chase studies have been taken to indicate that GTP within subunits in the microtubule is able to freely exchange with nucleotide in solution (Carlier & Pantaloni, 1981). We wish to determine the significance of such exchange into steady-state microtubules, relative to the diffusional and treadmilling paths for label incorporation. Such an analysis has been found to be feasible, as a result of the fact that GTP in the E site of tubulin subunits dissociates relatively slowly. This has allowed a comparison of the reactivity of t~bulin-[~H]GTP and [3H]GTP for label uptake in very brief pulse labeling experiments. Also, the slow dissociation of GTP has allowed analysis of tubulin-['H]GTP pulse/GTP chase experiments, in which the slow dissociation can influence the kinetics for the chase phase of the reaction.