Calcium and gadolinium ions stimulate the GTPase activity of purified chicken brain tubulin through a conformational change.
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 microM. 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-AIF4-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-Pi-tubulin from the microtubule ends is extremely slow compared with the rate of GDP-subunit dissociation, supporting the concept that the GTP- and GDP-Pi-tubulin cap at the ends of microtubules regulates their dynamic instability.
Soto
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Departamento de Biologiá, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Rodríguez
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Monasterio
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D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002645:Chickens; D005459:Fluorides / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D005682:Gadolinium / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D011487:Protein Conformation / Q000187:drug effects; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D017607:Aluminum Compounds; D014404:Tubulin; D005682:Gadolinium; D020558:GTP Phosphohydrolases; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride