THE CENTRAL PROBLEMS OE THE BIOCHEMISTRY OF CELL DIVISION 483 



hands of Dr. Katsuma Dan and myself [20], is to stabilize the structure 

 artificially, risking the distortion of some of its chemical properties but at 

 least obtaining it as a pure isolate for brute analysis. In effect, our earlier 

 methods — and the ones on which a good deal of our present information 

 depends — rested on the stabilization of the mitotic apparatus by immersing 

 dividing cells, usuallv sea urchin eggs, in 30",, ethanol at — 10 . F'ollowing 

 this stabilization, we could free and clean the mitotic apparatus by dispers- 

 ing the rest of the cell, which did not appear to be stabilized, with various 

 detergents and other dispersing agents. For the second step, we in our 

 laboratorv have most often used digitonin, although ATP and urea 

 (unpublished experiments with Dr. Rollin Hotchkiss) were also effective. 

 When I refer to results obtained by these methods, I shall generally 

 speak of the " alcohol-digitonin method ". 



A second and more demanding approach to a more natural isolation is 

 to attempt to mimic, in the isolation medium, the conditions in the cell 

 which provide for the stability of the mitotic apparatus. This could well 

 be hopeless according to the above-mentioned hypothesis of a dynamic 

 stabilitv. If the cell must be continuously active in some way to sustain the 

 structure, of the mitotic apparatus, then this activity could be mimicked 

 only if it were expressed in some simple terminal product or condition. 

 The approach to such a method depended on the hypothesis (discussed by 

 Mazia [25]) that the molecular interactions responsible for the structure of 

 the mitotic apparatus involved sulphur bonds and possibly S — S bonds. 

 On the basis of experience that will not be discussed here, it was imagined 

 that an intermolecular ( — SH)-(S- -S) equilibrium might be "poised" in 

 one direction or another by an appropriate SH or S — S reagent. To poise 

 it in the direction of S — S, we chose dithiodiglycol (OHCH.,CH.,S — 

 SCHoCHoOH). Whether or not the reasoning was correct — and one must 

 admit that it was somewhat woollv — this line of attack finally was success- 

 ful. The mitotic apparatus could be isolated in a medium consisting of 

 isotonic (i m) dextrose or sucrose, lo^^ m versene, and 0-15 M dithiodi- 

 glvcol at pH 6 -0-6 -3. An important point is that it is unstable if the 

 dithiodiglvcol is omitted and becomes unstable even after isolation if 

 this substance is removed. In our more recent work, the sucrose medium 

 has proved to be preferable to dextrose for the purpose of eliminating other 

 cvtoplasmic particles ; otherwise, I am not sure that it makes much 

 difference which sugar is used. 



Thus this method, which I will refer to as the DTDG method, is 

 comparable to those used for other kinds of particles with the exception of 

 the requirement for dithiodiglycol. It must be said that we have not yet 

 compared dithiodiglvcol with related compounds. The essential steps of 

 the isolation are these: (i) Sea urchin eggs are inseminated and immedi- 

 atelv transferred to a medium of Ca-free sea water, versene (o-oi m), and 



