The Mitotic Apparatus. Physical Chemical Characterization of the 22S Protein Component and Its Subunits

The major 22S protein of the hexylene glycol-isolated mitotic apparatus has been characterized from spindle isolates and extracts of whole eggs and acetone powders of eggs from the sea urchins Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Arbacia punctulata. The protein is free of nucleotide, lipid, and ATPase activity. Essentially identical in amino acid composition, proteins from these species show a relatively high content of glutamic and aspartic acids and are fairly rich in hydrophobic amino acids. Optical rotatory dispersion studies indicate a helical content of about 20%, a value consistent with the proline content of the protein. The purified proteins have sedimentation rates in the range of 22-24S, diffusion constants of 2.4-2.5F, intrinsic viscosities of 3.7-4.3 ml/g, a partial specific volume of 0.74, and an average molecular weight of 880,000. Electron microscopy indicates a globular molecule with dimensions of approximately 150 by 200 A; such size and symmetry are consistent with hydrodynamic measurements. The 22S protein yields 6-7S, 9-10S, and 13-14S subunits below pH 4 or above pH 11. The 13-14S component has an estimated molecular weight of 600,000-700,000. A 5-6S particle is formed in 8 M urea or 5 M guanidine hydrochloride, while at pH 12 the 6-7S subunit is seen; each particle has a molecular weight of 230,000-240,000. In 8 M urea plus 2% mercaptoethanol or at pH 13, the molecular weight becomes 105,000-120,000; under these conditions the particle sediments at 2.5-3S and 4S, respectively. On the basis of these molecular weights, the 6-7S, 9-10S, 13-14S, and the parent 22S particle should be dimer, tetramer, hexamer, and octamer, respectively, of the 105,000-120,000 molecular weight subunit. The various subunits will reform the 22S particle when returned to neutral buffer, with the exception of the mercaptoethanol-treated urea subunit where breakage of disulfide bonds results in a polydisperse aggregate. The 22S particle itself is not susceptible to sulfhydryl reagents, implying either that the disulfide bonds are inaccessible or that they are unnecessary for maintenance of tertiary structure once the 22S particle has formed from subunits.