136 BRITTON CHANCE 



transfer reactions, and to define the path by which energy is coupled to the reversal 

 of the electron transfer reactions. 



Singer : I would like to ask Dr. Chance why he seems to prefer the diaphorase 

 of the ketoglutaric oxidase complex as being the agent responsible for the reduction 

 of pyridine nucleotide rather than the one of the respiratory chain, since, in the 

 first place, it is commonly believed that there is a spatial separation between these 

 enzymes and, therefore, the mechanism of electron transport between these two 

 systems would not be very obvious. In the second place, the partial inhibition by 

 amytal presents another difficulty, since the ketoglutaric system is not known to be 

 amytal-sensitive. 



Chance: I completely agree with Dr. Singer, but wanted to point out that 

 there are flavins which had been demonstrated to reduce DPN. 



P'renkel : Can you tell from your difTerence spectrum whether the DPN is free 

 or enzyme-bound ? Dr. Kaplan has informed me that the reduction of enzyme- 

 bound DPN may require appreciably less free energy than the reduction of free 

 DPN. 



Chance : That is a very interesting observation. The intra-mitochondrial DPNH 

 is bound but it requires about two ATPs for each DPN reduced so considerable 

 energy is required. Its potential may indeed be higher than —300 millivolts but its 

 surely not zero. 



Arnon : I would still like to come back to Dr. Chance's comment on the possible 

 significance of this reaction in non-photosynthetic cells. 



Chance: I thought I had answered that question. This morning Prof. Lindberg 

 referred to some very interesting possibilities where thyroxine might interact with 

 the reduction of DPN and I think this is certainly an example of how this would be 

 a pathway of production of reducing power in the cell, which could be under 

 hormonal control. 



Slater: Dr. Chance's explanation of his results on the basis of reversal of the 

 respiratory chain is very feasible. I am not completely sure that that is the only 

 possible explanation of his results, but this is something I do not think we can 

 possibly go into now. My first question follows on from what Dr. Singer asked. 

 Which flavoprotein do you think you are studying when you are following the 

 flavoprotein spectrum ? The second question relates to the very interesting anaero- 

 bic experiment with dithionite and ATP where you get DPN reduced and cyto- 

 chrome c oxidized ; what is the stoicheiometric relationship between the amount of 

 DPN reduced and cytochrome c oxidized ? 



Chance: The answer to the first question is that we don't know for sure, 

 because the flavins are unfortunately summed by measurement at 465 m/x, but the 

 amount of flavin which is involved in this pathway is the major portion of the 

 flavin which one observes spectroscopically. Two types of answer are available 

 to your second question. We added 5 -6 /xmoles ATP and we found a total oxidized 

 ^3, a, c and flavin of i -5 one-electron equivalents to be oxidized. In other experi- 

 ments the DPN reduction was slightly in excess of the cytochrome oxidation, but 

 we have not included Q oxidation because it is hard to assay it quantitatively when 

 we add ATP. We find that DPN is reduced faster and flavin oxidized faster than 

 cytochrome c. This is apparently the couple into which energy can be put most 

 easily. 



