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Stoichiometry of GTP hydrolysis and tubulin polymerization

R Maccioni, N W Seeds

    Proceedings of the National Academy of Sciences of the United States of America
    |February 1, 1977
    PubMed

    Insights

    Guanosine triphosphate (GTP) is essential for microtubule formation from purified tubulin. GTP analogs cannot substitute for GTP, and its hydrolysis is coupled to tubulin incorporation into microtubules.

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Background:

    • Microtubules are crucial cytoskeletal components involved in various cellular processes.
    • Tubulin, the protein subunit of microtubules, requires guanosine triphosphate (GTP) for polymerization.

    Purpose of the Study:

    • To investigate the specific role and requirements of GTP in microtubule formation using purified lamb brain tubulin.
    • To determine the stoichiometry of GTP hydrolysis during tubulin polymerization.

    Main Methods:

    • Purification of lamb brain tubulin using affinity chromatography.
    • Assessment of microtubule polymerization kinetics using a rapid filter assay.
    • Analysis of GTP concentration dependence and inhibition by nucleotide analogs.

    Main Results:

    • Guanosine triphosphate (GTP) is indispensable for maximal microtubule polymerization rate and extent.
    • GTP analogs (guanylylmethylenediphosphate, guanylylimidodiphosphate) cannot substitute for GTP.
    • GDP and guanylylimidodiphosphate act as competitive inhibitors of GTP polymerization.
    • A stoichiometry of two GTP molecules hydrolyzed per tubulin dimer incorporated into microtubules was determined.

    Conclusions:

    • GTP is a critical requirement for microtubule assembly from purified tubulin.
    • GTP hydrolysis is stoichiometrically coupled to tubulin incorporation into the growing microtubule polymer.

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