488 daniel mazia 



(a) bonding of the mitotic APPAR.\TUS 



So far as the mitotic apparatus is concerned, the present picture is 

 confusing, although not in an unconstructive way. The original design of 

 an isolation method by Dan and myself was based on an hypothesis that 

 S — S bonds were involved in the polymerization of macromolecules into 

 a coherent mitotic apparatus. We proceeded first in a seemingly strange 

 way, artificially stabilizing the mitotic apparatus by deliberately making 

 more S — S bonds by oxidation with peroxide, but it did work. When a 

 method was developed which avoided the use of such an oxidizing agent, 

 the isolated mitotic apparatus seemed to be an S — S bonded structure, 

 soluble only by methods which reduce such bonds [30]. Then this turned 

 out to be a partial oxidation artifact, for it was discovered by Dr. Zimmer- 

 man in our laboratory that the freshly isolated apparatus which had been 

 given no chance to oxidize could be dissolved by salyrgan and/)-chloromer- 

 curibenzoate. At the same time, Kawamura and Dan [36] showed by 

 cytochemical means that the mitotic apparatus during the stages at which 

 it was forming was strikingly rich in protein-SH, which became less 

 prominent during the terminal stages of mitosis. It would be satisfactory, 

 and would meet all the facts, to suppose the following: (i) That the 

 apparatus contains SH groups in closely apposed pairs, easily oxidized but 

 not necessarily existing as S — S links in the living condition, and (2) That 

 a linkage of unknown character between these vicinal SH groups holds 

 the molecules together in the structure of the mitotic apparatus. Such a 

 link might be split by agents such as salyrgan and PCMB. There is in- 

 direct evidence for the occurrence of such thiol, non-S — S, linkages in 

 other situations (reviewed by Jensen [41]) but the nature of the bond is 

 unknown. I take it that most chemists are not happy with the idea of 

 hydrogen bonding through SH, although it has been defended. In general, 

 the idea that intermolecular hydrogen bonding is prevalent in the mitotic 

 apparatus has been an appealing one (e.g. Gross [42]) and it has seemed to 

 some that an hypothesis of extensive S — S bonding was not consistent 

 with the instability of the mitotic apparatus. Thus, the situation was that 

 the hypothesis of an S — S bonded system led to certain positive results 

 but tests of the hypothesis always favoured the implication of thiol groups 

 in some other way. It was a looser hypothesis concerning sulphur bonding 

 that led to the development of the DTDG method of isolating the mitotic 

 apparatus in a more native condition. The speculations involved will be 

 discussed in a later section. 



(b) the thiol cycle 



It should have been stressed earlier that the instability of the mitotic 

 apparatus is not merely an inconvenience for its isolation ; it is a funda- 



