General Discussion 305 



the rat thymus is rather particular in that the nucleoprotein is not 

 completely dissociated in this salt solution. If you take the same 

 thing with beef thymus you get a much greater degree of dissociation 

 and you don't get this remarkably high specificity. 



Haddow: Dr. Howard has put forward a suggestion, namely that 

 Dr. Dale should tell us to what extent he believes his five questions 

 have been answered. 



Dale: In my presentation, I deliberately did not express any 

 opinion, giving simply a background survey, and at the end of it I 

 put some questions (p. 33) which involved the two main manifesta- 

 tions of indirect action, the dilution effect and the protection effect, 

 and asked this audience whether there is scope for these two mani- 

 festations to explain the possible mode of action of enzymes. One of 

 the main questions, i.e. the fourth question, was answered to a cer- 

 tain extent in the way in which I put it, but Prof. Krebs in his con- 

 tribution modified this and answered it more or less in this way, 

 that he thought the determining factor is not so much the disturb- 

 ance of the enzyme itself, but of the substance which is concerned 

 with the enzymatic process, that is of the substrate ; and of course the 

 dilution effect will just as well act on that if it acts at all. 



I understood from Prof. Kreb's answer to my .third question that 

 there is a possibility of a minute amount of available substrates being 

 interfered with at the steady-state, which may be low. The steady- 

 state concentration of the substrates may be a determining factor, 

 and may be responsible for disorganization of consecutive steps in 

 the enzymatic action. 



I also asked the question "Are these intermicellar spaces in the 

 inhomogeneous cell structure filled with high concentrations of pro- 

 tective substances?" This is an objection that is usually made, that 

 there are plenty of protective substances which will obviate the 

 indirect action. Now there are model experiments by Stein and one 

 co-author in which he irradiated gelatin gel in which he had incorpor- 

 ated methylene blue, and there was little or no interference with the 

 action of degradation of this methylene blue. In this case one would 

 say, taking the gelatin as the cytoplasmic model for the interior of 

 the cell, that the micellar structure of this gel did not interfere with 

 the indirect action. Indirect action could still take place in the 

 solvent-filled spaces between those gelatin structures. Also, Gordon 

 and his co-worker used agar gels and did not find a protective effect 

 of these on substances distributed in these gels. In answer to my 

 fifth question, I would say quite emphatically that any experi- 

 ments on these lines are doomed from the start. They cannot 

 give any indication of what is happening, because all that this type 

 of experiment is concerned with is the total amount which is present. 



