THE MITOTIC APPARATUS Physical-Chemical Factors Controlling Stability

Previous investigations have shown that the mitotic apparatus (MA) can be isolated from dividing sea urchin eggs in water buffered at pH 5.6 and that the addition of 1 M hexanediol to the solution raises the usable pH to 6.4. Long chain glycols appeared to be much more effective than related compounds in increasing the stability of the MA, and the aim of the investigations reported here was to determine the basis of this specificity. These experiments show that this impression of specificity is misleading and that under suitable experimental conditions a variety of compounds can be substituted for the glycols. A number of alcohols will duplicate the action of the glycols in stabilizing the MA at pH 6.4, but they must be used at a similar per cent concentration rather than at a similar molar concentration. Increases in the concentration of alcohol or glycol allow isolation at more alkaline pH values, and a pH-concentration relation for the stability of the MA, covering the range from pH 5.6-8, has been determined. These results indicate that the action of these compounds in stabilizing the mitotic apparatus is non-specific and is similar to their effects on the solubility of proteins. The isolation and stabilization of the mitotic apparatus can thus be viewed as a function of the solubility properties of its constituent proteins, opening a variety of new experimental approaches to this problem. I N T R O D U C T I O N More than ten years have passed since Mazia and Dan (I 3) first demonstrated that the intact mitotic apparatus could be isolated from dividing sea urchin eggs. In the intervening years, new methods of isolation have been developed and the structure and properties of the isolated MA investigated in some detail (4, 7, 8, 10, 12, 14-16, 19). The factors which control the stability of the MA are still poorly understood, however, as the development of isolation methods has been largely empirical and no general principles have emerged. Since some understanding of the stability properties of the MA in vitro seems a necessary preliminary to an attack on such problems in vivo, a more systematic study of the isolation process is clearly required. The object of such an investigation should be to define, in the most general terms possible, the conditions necessary for the isolation of the mitotic apparatus, thus allowing the formulation of testable hypotheses concerning the mechanism involved in the stabilization of the MA in vitro. The original method of isolation developed by Mazia and Dan (13) was based on the extended treatment of metaphase eggs in a solution of 30 per cent ethanol at -10°C, after which the cytoplasm was dispersed, freeing the intact mitotic apparatus. This method was sensitive to both concentration and temperature. At lower alcohol concentrations the MA disintegrated, while at higher concentrations the cytoplasm of the cell coagulated. Similar 137 on A ril 3, 2017 D ow nladed fom Published April 1, 1965