Stoichiometry of GTP hydrolysis and tubulin polymerization

Abstract

Microtubule formation from lamb brain tubulin isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethylenediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of microtubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 10(4) M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization. Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found.