THE CENTRAL PROBLEMS OF THE BIOCHEMISTRY OF CELL DIVISION 485 



mitotic apparatus, suspended in a smooth homogenate of cytoplasm. 

 (6) The mitotic apparatus may readily be purified and washed by very 

 low speed (200 500 g) centrifugation. Fortunately, most of the smaller 

 particles remain suspended in the dense sucrose medium. 



The following properties of the mitotic apparatus as isolated bv the 

 DTDG method are pertinent and interesting: 



(i) They are extremely sensitive to Ca and J\Ig, becoming irreversibly 

 stabilized. A Ca"^ concentration of 5 x 10"^ m suffices to stabilize them. In 

 this form they are beautiful to behold (Fig. 2) because the fibres become 

 highly condensed, but we cannot dissolve them for further chemical work. 



(2) They are osmotically sensitive, swelling and shrinking as the 

 sucrose concentration is varied. They disperse at low sucrose concentrations. 



(3) They may be dissolved in a number of ways if they have not " seen " 

 Ca++ or Mg+^. For enzvme studies we dissolve them in isotonic (0-53 m) 

 KCl at pH 8. This has the advantage that the osmotically sensitive yolk 

 particles which are a major contaminant are not lysed and can be separated 

 by centrifugation, along with other particles, probably ribosomes, that are 

 known to be embedded in the structure [22]. 



It would be brash to suggest that this isolation represents achievement 

 of the goal of obtaining a fully natural mitotic apparatus. A more limited 

 objective was to obtain the mitotic apparatus in a form suitable for studies 

 of enzyme activity, especially of enzymes concerned with ATP. The 

 methods employing alcohol and detergents did not preserve such activitv ; 

 while the DTDG method does. A second objecti\e, and indeed a long-term 

 ideal of these studies, was to obtain the isolated mitotic apparatus as an 

 effective "model" in the Weber [26] sense of the term. So far, this has 

 failed. We have found no conditions imder which the isolated mitotic 

 apparatus will mo\e chromosomes. 



6. Survey of the chemistry of the isolated mitotic apparatus 



The alcohol-digitonin method provided clean preparations of mitotic 

 apparatus which retained the essential and expected morphological 

 features, and which could be regarded as suitable for the study of some of 

 the major structural macromolecules. Much of the information that has 

 been obtained has already been reviewed, and I shall only list the findings. 



I. The mitotic apparatus isolated by the old method consists largelv 

 of protein. Conjugation of RNA to the protein has been studied in some 

 detail, but comparable studies have not been made on conjugation of lipid 

 or carbohydrates. There seemed to be little point in analvzing for lipids 

 after isolation with digitonin, and we have tended to formulate the struc- 

 tural problems pretty much in terms of protein chemistrv. In view of 

 electron microscopic evidence describing the filaments of the mitotic 



