On the relationship between nucleotide hydrolysis and microtubule assembly: Studies with a GTP-regenerating system

The assembly of pure tubulin dimer has been studied in two buffer systems (containing low and high glycerol/Mg), using a regeneration system protocol to assess the amount of GDP-tubulin in the assembling polymer. For both assembly systems studied, the GDP content is effectively stoichiometric with tubulin throughout assembly. This indicates a high degree of coupling between assembly and GTP-hydrolysis, giving a hydrolysis rate at least 10-fold faster than previously deduced. The steady state GTP hydrolysis rate is quantitatively consistent with this finding. We conclude that the extent of any GTP-tubulin cap is below the detectable limit, both during elongation and at steady state. MT-protein; microtubule protein; MAPs; microtubule associated proteins; PC-tubulin; tubulin dimer from phosphocellulose chromatography; GTP- and GDP-tubulin denotes the nucleotide at the exchangeable nucleotide binding site (E-site)RS; enzymic GTP-regenerating system.