Temperature jump relaxation study of microtubule elongation in the presence of GTP/GDP mixtures.

GDP was added to microtubules at steady state. The amount of dissociation obtained was dependent on the GDP/GTP ratio and a method was developed to extrapolate to pure GDP conditions. From this extrapolation it was concluded that in the absence of GTP no elongation events occur. It was shown that at 35 degrees C nucleotide exchange is very fast, but at 25 degrees C, it is rate limiting for GDP-induced dissociation. Relaxation experiments, using temperature jumps before and after the addition of GDP, show that the nucleotide composition of the ends has to be taken into account. The model accepted so far cannot explain the observations. Several model mechanisms are described and their implications for equilibrium and relaxation data are analysed. All the acceptable models predict an increase in treadmilling efficiency at high GDP concentrations.