The mitotic apparatus isolated in glycerol containing medium

The mitotic apparatus of the sea urchin egg was isolated at 30°C in an isolation medium containing glycerol, which is known to stabilize microtubules. After isolation in the 1 M glycerol isolation medium, the mitotic apparatus was stabilized on addition of glycerol to a final concentration of 3 to 4 M. Without the addition, the chromosomes were disjointed from the spindle, and the interzonal region between separating chromosomes was fragile resulting in separation of half spindles. Lowering the temperature of the isolation medium to 20°C or below, the procedure allowed the isolation of spindles. The isolated spindle behaved in a manner similar to the mitotic apparatus, from the effect of glycerol concentration. The glycerol mitotic apparatus contained tubulin which was extractable with the isolation medium containing Ca ions or an organic mercurial. Tubulin was also extracted upon lowering the temperature to 0°C in the presence of guanosine triphosphate. Addition of KCl to a final concentration of 0.6 M immediately dispersed the mitotic apparatus. The extract revealed a colchicine binding of 0.011 mole per 105,000 X g of protein. The colchicine binding complex was found to have a molecular weight of 105,000. Diethylaminoethyl Sephadex column chromatography of the KCl extract allowed the elution of the tubulin fraction which bound 0.1 mole colchicine per 105,000 X g of protein. The mitotic apparatus tubulin was shown to contain α and β subunits with mobilities quite identical with those of brain tubulin subunits. The molecular weights of the α and β subunits were 55,000 ± 1,000 and 51,000 ± 1,000, respectively.