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J. Biol. Chem., Vol. 282, Issue 8, 5570-5581, February 23, 2007

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Kinetic Analysis of Tubulin Assembly in the Presence of the Microtubule-associated Protein TOGp^*

Claude Bonfils¹, Nicole Bec, Benjamin Lacroix, Marie-Cécile Harricane, and Christian Larroque

From the INSERM, EMI 229, CRLC Val d'Aurelle, 34298 Montpellier, France and Centre de Recherche en Biochimie Macromoléculaire, CNRS, 1919 route de Mende, 34293 Montpellier, France

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.

Received for publication, June 13, 2006 , and in revised form, November 14, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: INSERM EMI 229, CRLC Val d'Aurelle-Paul Lamarque, Rue des Apothicaires, 34298 Montpellier cedex 5, France. Tel.: 33-4-67-61-85-36; Fax: 33-4-67-61-37-87; E-mail: bonfils{at}valdorel.fnclcc.fr.

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