The Isolation and Biochemical Characterization of the Mitotic Apparatus of Dividing Cells

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Abstract
The term mitotic apparatus (MA) includes spindles, asters, centrioles, nuclei (before breakdown of the nuclear membrane) and chromosomes (after breakdown of the nuclear membrane). Eggs of sea urchins Strongylo-centrotus franciscanus and S. purpuratus were used, and fertilization membranes removed by treatment with a mixture of crystalline trypsin and chymotrypsin. The eggs were inseminated, allowed to develop in rather dilute populations, then concentrated by centrifugation. The MA was preserved by immersing the eggs in 30% ethanol at about -10[degree]C and was separated from the cytoplasm mechanically or by solubilization in the presence of H2O2 and Duponol D. Cytological observations were made with phase contrast microscopy. The whole MA seemed to behave as a single physical and chemical unit in the isolation procedure. By following the MA through all stages from the living to the final isolated product, it was clear that the major structures such as spindle fibers and astral rays were not artifacts, although they might have been modified in the process. The isolated MA showed positive birefringence along the spindle fiber axis as in the living cell. A protein was isolated from the MA by isoelectric precipitation at pH 6.0.Detns. of the protein content of Ma indicated that the protein accounted for most of the mass of the MA and for approx. 2% of all the protein of the egg. U-v. absorption curves of the MA protein were like those of typical proteins. The MA protein gave a single boundary in the ultra-centrifuge, with an estimated particle wt. of approx. 45,000. When the MA isolated by the peroxide-detergent method was treated with 5% sodium thioglycollate soln. either before or together with a 1% detergent soln., the MA was dissolved completely by the detergent, indicating that the stabilizing effect of S-S linkages introduced with H2O2 was reversed by reducing the S-S groups.

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