170 MACROMOLECULAR COMPLEXES 



mitotic apparatus. They shared similar molecular weights; each 

 contained a ribonucleotide component; they were both relatively 

 asymmetrical molecules; and the calcium-insoluble fraction repre- 

 sented about 10 per cent of the total protein of the unfertilized egg. 

 Mazia and Roslansky (1956) have estimated that the cell invests 

 about 12 per cent of its total protein in the mitotic figure. This im- 

 mediately gave rise to the speculation that the calcium-insoluble 

 fraction was the long-sought-after precursor component, and now 

 we had at our disposal the means to test this more directly The 

 calcium-insoluble fraction could be purified to the extent that, when 

 analyzed by gel-diffusion methods, it gave rise to only one distinct 

 band in reaction with antiserum to unfertilized egg antigens. Thus 

 it was a simple matter to see if the precursor- 1 component band was 

 the same as the only band assignable to the calcium-insoluble frac- 

 tion. 



Figure 2 summarizes the essential results. When dissolved mi- 

 totic apparatus and calcium-insoluble fraction are each compared to 

 a solution of unfertilized egg antigens, one observes that the band 

 associated with the dissolved mitotic apparatus merges with the one 

 assignable to the unfertilized egg antigens, but it is readily seen that 

 this is not the same band that merges with the only one attributable 

 to the calcium-insoluble fraction. This clearly demonstrates that the 

 calcium-insoluble fraction bears no direct structural relationship 

 to the mitotic apparatus and demolishes the attractive possibility 

 that it represents the precursor protein. Although there can be little 

 doubt about the existence of the precursor- 1 component, it could not 

 be identified with the calcium-insoluble fraction, which was the only 

 protein fraction obtained in a homogeneous form from unfertilized 



eggs- 



The precursor- 1 component was invariably present in all solutions 

 of mitotic apparatus isolated from preserved material by the digi- 

 tonin method. These solutions were obtained by bringing an 

 aqueous suspension of mitotic apparatus to pH 10-10.5. At these 

 pH values, no mitotic figures were recognizable under phase con- 

 trast, while the contaminating cytoplasmic particles remained rela- 

 tively unaff^ected by the treatment. On the other hand, pH values 

 in the range 8.5-9.0 were adequate to dissolve completely the 

 mitotic figures isolated directly from living material, while leaving 

 many of the undesirable cytoplasmic particles intact. These could 

 be removed by centrifugation. Such solutions also gave rise to the 



