Assembly of microtubule protein: role of guanosine di- and triphosphate nucleotides

Abstract

A quantitative analysis of the interplay between GTP and GDP in [pig brain] microtubule assembly and accompanying GTP hydrolysis was performed when tubulin was polymerized in the presence of microtubule-associated proteins (MAP) which display an interfering GTPase activity. The use of adenylyl .beta.-imidodiphosphgate, which specifically inhibits the MAP GTPase activity, and of vinblastine (or podophyllotoxin), which specifically inhibits GTP hydrolysis due to tubulin, made possible a study of the exclusive GTP hydrolysis associated to microtubule assembly. GDP binds to microtubule ends with an affinity comparable to GTP, thus strongly inhibiting both the elongation process and the steady-state GTP hydrolysis at microtubule ends. GDP shifts the equilibrium between tubulin and microtubules toward disassembly. The MAP which are released from the microtubules during the GDP-driven depolymerization cluster on the remaining microtubules. The resulting increased stability of microtubules is quantitatively consistent with the the decrease in the critical concentration of the polymerizing species GTP-tubulin.