Role of the dianionic form of the GTP gamma-phosphate in the polymerization process of tubulin.
To determine whether tubulin polymerization requires the bivalent metal-GTP complex with the gamma-phosphate in the dianionic form, the effect of GTP(gamma F) on the polymerization process was studied, in the presence of either magnesium or manganese. P3-fluoro P1-5'-guanosine triphosphate (GTP(gamma F)) was a competitive inhibitor (Ki = 1.8 X 10(-4) M) of the GTPase activity of tubulin-colchicine complex, stopped the polymerization process during the course of reaction and no depolymerization occurred. This indicates that GTP(gamma F) has access only to the nucleotide exchangeable site of the free tubulin dimer. Tubulin has one mole of magnesium tightly bound per mole of dimer. In order to know whether the inhibitory effect of GTP(gamma F) was due to the release of the metal, magnesium was replaced for manganese (a paramagnetic ion) and the paramagnetic effect of manganese on the fluorine NMR signal from the GTP(gamma F)-tubulin-metal complex was followed. Longitudinal and transversal relaxation rates measurements of the 1 degree F-NMR signal allowed to determine that the upper distance from the manganese site to the fluorine atom was between 6 and 8 A. These studies demonstrate that the dianionic form of the terminal phosphate of the metal-GTP complex, at the nucleotide exchangeable site, is essential to stimulate tubulin polymerization.
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013873:Thionucleotides / Q000378:metabolism*; D014404:Tubulin / Q000096:biosynthesis*
D046911:Macromolecular Substances; D013873:Thionucleotides; D014404:Tubulin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D006160:Guanosine Triphosphate; D008274:Magnesium