In Vitro Polymerization of Marine Egg Tubulin into Microtubules

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Abstract
Marine egg tubulin could be purified from unfertilized or fertilized sea urchin [Hemicentrotus pulcherrimus and Anthocidaris crassispina] and starfish [Asterias amurensis] eggs by the use of DEAE-Sephadex A-50 ion exchanger. Of the total protein in the 20,000 g egg extract supernatant .apprx. 0.2% was recovered as DEAE-Sephadex purified tubulin which assembled into microtubules upon incubation at 35.degree. C. Applying the routine polymerization-depolymerization procedure for tubilin purification to the initial egg tubilin 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 MW of 110,000 as determined by gel-filtration or of 128,000 as determined by the sedimentation-equilibrium method. Egg tubilin dimer possessed binding activities of 0.8 mol colchicine and 0.80-0.97 mol exogenous 3H-GTP at the exchangeable site. Its .alpha. and .beta. subunits showed the same mobilities as those of porcine brain tubulin or outer fiber tubulin of sea urchin sperm flagella on SDS [sodium dodecyl sulfate]-polyacrylamide gels. When the egg tubilin fraction was warmed at 35.degree. C, microtubules were reconstituted in parallel with increase in viscosity. Microtubule-depolymerizing agents such as Ca2+, low temperature, colchicine or SH reagents inhibited in vitro polymerization of egg tubulin and induced depolymerization of reconstituted egg microtubules. The purified egg tubilin fraction polymerized into microtubules by itself, the assembly being strikingly stimulated by the addition of exogenous nuclei fractions for polymerization.

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