Calcium and Gadolinium Ions Stimulate the GTPase Activity of Purified Chicken Brain Tubulin through a Conformational Change

Abstract

Ca²⁺ and Gd³⁺ stimulated the GTPase activity of chicken brain tubulin 13- and 26-fold, respectively. Mg²⁺, Tb³⁺, and Na⁺ had no effect. This GTPase activity showed a saturation behavior with Ca²⁺ and Gd³⁺ with a maximal activity of 0.26 ± 0.026 and 1.15 ± 0.78 nmol min^-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 ± 0.18 and 0.011 ± 0.007 mM, respectively. In the presence of Ca²⁺, the GTPase activity was proportional to tubulin concentration in the range 0.9−31.8 μM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca²⁺ were 0.71 ± 0.1 and 0.049 ± 0.043 mM, respectively. The similarity of the Ca²⁺ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not directly involved in the mechanism of inhibition of Ca²⁺-dependent tubulin assembly. This inhibition could be better explained by the formation of a nonfunctional conformational state of tubulin induced by Ca²⁺ that is responsible for the GTPase activity. Quenching of the intrinsic fluorescence of tryptophan induced by Ca²⁺ showed an apparent dissociation constant of 0.14 ± 0.005 mM, in the range of values determined through tubulin polymerization inhibition or through the induction of GTPase activity by Ca²⁺. Acrylamide-induced quenching of the intrinsic fluorescence showed values of the Stern−Volmer constants of 5.4 ± 0.12 and 5.0 ± 0.15 M^-1 in the absence and presence of Ca²⁺, respectively. These results support the hypothesis that the inhibition of tubulin polymerization and the induction of the GTPase activity by Ca²⁺ is mediated by a conformational change. Ca²⁺ failed to induce depolymerization of GDP−AlF₄- microtubules; this could be explained by a model in which Ca−tubulin is unable to assemble into microtubules and the rate of dissociation of GDP−P_i−tubulin from the microtubule ends is extremely slow compared with the rate of GDP−subunit dissociation, supporting the concept that the GTP− and GDP−P_i−tubulin cap at the ends of microtubules regulates their dynamic instability.