84 Discussion 



Krebs : But it becomes stable when it is attached to the ~. 



Slater: One determines DPN and DPNH, but more total DPN is 

 found after incubating mitochondria. 



Krebs: That may mean either that more is liberated or inore is 

 available, or that some of it has been destroyed in the initial material. 

 In that case it would mean that it would stabilize the initial material. 

 If in your ordinary determination you had some losses, you would get a 

 certain lower value than you would get if you did not have these losses 

 and, therefore, the bound DPN would prevent losses, if it were more 

 stable. 



Slater: These procedures can be controlled and we do not get losses 

 with DPN or DPNH. The recoveries of DPN and DPNH are quantita- 

 tive. 



Potter: Prof. Slater, when you carry out the incubation of mito- 

 chondria, do you then centrifuge them down and treat the pellet with 

 acid or alkali, as the case may be, or do you work on the whole incubation 

 mixture? 



Slater: We work on the whole incubation mixture. 



Potter : So it is the sum of what is extractable from the mitochondria 

 plus what may have leaked out into the medium. 



Slater: Yes, it is the total. The rather thick mitochondrial suspension 

 in sucrose is deproteinized at various times. 



Racker : How do you explain that TPN '---I goes up? Do you visualize 

 this as a transfer reaction from DPN-^I to TPN'~-^I? 



Slater: Yes, that would be one possibility. However, Dr. Purvis has 

 not yet studied the effects of amytal or of substrate on TPN~I. These 

 are the substances which cause DPN '^I to go up. Therefore, the forma- 

 tion of extra TPN '--'I has not yet been demonstrated in the same way as 

 the formation of extra DPN-^I. What has been established is that 

 incubation of mitochondria with ADP, phosphate or DNP, causes the 

 appearance of extra TPN as well as of extra DPN. We interpret this in 

 terms of a breakdown of TPN^I as well as of DPN^I. TPN^I could 

 be formed by reaction of DPN~I with TPN, a sort of "pyridine nucleo- 

 tide trans-I-ase" reaction, as you suggest. 



Hess: What is the stoichiometrical relationship between the ADP 

 that you added and the amount of DPN^I which appeared? Also, I 

 would like to know whether it is excluded that there is a de novo syn- 

 thesis of DPN. 



Slater: The extra DPN appears very quickly — within a few minutes. 

 This occurs in the presence of ADP, inorganic phosphate or DNP, but 

 not ATP. If something were being synthesized, the opposite result 

 might be expected; one would expect to get it synthesized in presence 

 of ATP, but certainly not in presence of DNP. Although DPN is not 

 supposed to be synthesized in mitochondria, but in the nuclei, this 

 would not by itself be conclusive since there might be another unsus- 

 pected system for the synthesis of DPN in the mitochondria. Our 

 reason for thinking that this is not the case is that the formation of 

 DPN does not take place with ATP whereas it does with DNP. The 

 process has all the characteristics of a breakdown of a compound con- 



