In Vitro polymerization of marine egg tubulin into microtubules

Marine egg tubulin could be purified from unfertilized or fertilized sea urchin and starfish eggs by the use of DEAE-Sephadex A-50 ion exchanger. About 0.2% of the total protein in the 20,000 g supernatant of the egg extract was recovered as DEAE-Sephadex purified tubulin which assembled into microtubules upon incubation at 35°C. Applying the routine polymerization-depolymerization procedure for tubulin purification to the initial egg tubulin preparation, almost pure egg tubulin could be obtained.The purified egg tubulin fraction was shown by analytical centrifugation to consist of only a 6.3S component, having a molecular weight of 110,000 as determined by gel-filtration or of 128,000 as determined by the sedimentation-equilibrium method. Egg tubulin dimer possessed binding activities of 0.8 mol colchicine and 0.80-0.97 mol of exogenous ₃H-GTP at the exchangeable site. Its α and β subunits showed the same mobilities as those of porcine brain tubulin or outer fiber tubulin of sea urchin sperm flagella on SDS-polyacrylamide gels.When the egg tubulin fraction was warmed at 35°C, microtubules were reconstituted in parallel with the increase in viscosity. Microtubule-depolymerizing agents such as Ca²⁺ ions, low temperature, colchicine or SH reagents all inhibited in vitro polymerization of egg tubulin as well as inducing depolymerization of reconstituted egg microtubules. The purified egg tubulin fraction polymerized into microtubules by itself, the assembly being strikingly stimulated by the addition of exogenous nuclei fractions for polymerization.