Stoichiometry of GTP hydrolysis and tubulin polymerization.

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Abstract
Microtubule formation from lamb brain tubulin isolated by affinity chromatography and freed of exchangeable nucleotide required GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethylenediphosphate and guanylylimidodiphosphate failed to replace GTP; neither the presence of microtubule associated proteins nor 5 M glycerol relieved the GTP requirement. The relation of GTP concentration and microtubule formation showed an association constant K = 1 .times. 104 M-1; GDP and guanylylimidodiphosphate were competitive inhibitors of GTP for polymerization. Using a rapid filter assay for microtubule formation that allowed the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of 2 molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules was found.