Reversible dissociation of the alpha beta dimer of tubulin from bovine brain

Abstract

The reversible, concentration-dependent dissociation of the alpha beta dimer of bovine brain tubulin (purified by phosphocellulose chromatography) has been demonstrated by equilibrium ultracentrifugation. The dissociation constant is approximately 8 X 10(-7) M at 4.6 degrees C in PM buffer (0.1 M piperazine-N, N'-bis(2-ethanesulfonic acid), 2 mM ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetic acid, 1 mM MgSO4, 0.1 MM guanosine triphosphate, 2mM dithioerythritol, at pH 6.9). This result was confirmed by observation of an appropriate dependence of the sedimentation coefficient of very dilute (is less than 0.5 mg/mL) tubulin on its concentration. Small zone gel filtration experiments on Bio-Gel P-150 also demonstrated an increase in peak elution volume with decreasing column load concentration. Reversibility of the dissociation was demonstrated directly by sedimentation velocity and gel filtration ion experiments on tubulin reconcentrated from dilute solution by pressure ultrafiltration. Control experiments accompanying the sedimentation equilibrium experiments showed that this tubulin retained, under the conditions of the experiments, both its ability to form microtubules and more than 70% of its initial colchicine-binding activity.