Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp.
The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.
Bonfils
Claude
C
INSERM, EMI 229, CRLC Val d'Aurelle, 34298 Montpellier, France. bonfils@valdorel.fnclcc.fr
Bec
Nicole
N
Lacroix
Benjamin
B
Harricane
Marie-Cécile
MC
Larroque
Christian
C
D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009369:Neoplasms / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate