Discussion 183 



DISCUSSION 



Chance: Prof. Dickens, with reference to your Table VI which 

 showed the DPNH/DPN and TPNH/TPN ratios in mitochondria, did 

 you treat the mitochondria with substrate prior to analysis or did they 

 contain only endogenous substrate? 



Dickens: They were not treated with substrate. 



Chance: Disruption of the mitochondria during analysis apparently 

 causes some oxidation of reduced pyridine nucleotide by activation of 

 the respiratory chain. Apparently this occurs just as you add per- 

 chlorate to stop the reaction. It also appears that the intramitochondrial 

 DPNH can be oxidized without affecting TPNH to the same degree. 

 This may be due to the fact that transhydrogenase activity is rather 

 low, the TPNH has not had time to follow the DPNH. 



Dickens: We found low transhydrogenase activities in liver too, but 

 Prof. Slater has stated earlier in this symposium (p. 13) that Dr. Purvis 

 in his laboratory has found reasonably adequate transhydrogenase 

 activity. With regard to the oxidation during the preparation, I 

 suppose that is a possibility. You mean that DPNH would be oxidized 

 preferentially, and the TPNH remain? 



Chance: DPNH can be rapidly oxidized by the isolated respiratory 

 chain. 



Dickens: It would be very difficult on this basis to explain the fact 

 that when the whole tissue is taken out of the animal and rapidly 

 dropped into boiling acid or alkali, as the case may be, very much the 

 same distribution of oxidized and reduced coenzymes is obtained; i.e. 

 in the whole tissue the total DPN is about 70 per cent or more oxidized, 

 while the TPN is nearly all present as TPNH. 



Glock: There is, in fact, about the same proportion of oxidized to 

 reduced form of each of the pyridine nucleotides in the whole tissue as 

 in the isolated mitochondria. 



King : In view of the fact that the pentose cycle enzymes exist almost 

 exclusively in the soluble, non-mitochondrial fraction, I wonder whether 

 there is another route for the direct oxidation of TPNH in the soluble 

 fraction. 



Dickens: It was first shown by Drs. Glock and McLean that these 

 enzymes are mainly in the soluble fraction of the cell. Reduced TPN 

 is therefore presumably mainly produced by this system in the soluble 

 part, and yet the mitochondria are full of reduced TPN. This is puzzling. 

 As regards the oxidative systems in the soluble part, they must be 

 comparatively inefficient too, because otherwise the whole TPN in the 

 cell would not be mainly in a reduced state, which it is. 



de Duve : Some observations made by Dr. Hers in my laboratory seem 

 to confirm the different state of oxidation of the two coenzymes. The 

 system studied by Dr. Hers is the conversion of glucose to fructose 

 in seminal vesicles. This appears to go through sorbitol by way of a 

 reduction which uses TPNH as an electron donor, and a subsequent 

 oxidation which uses DPN as an electron acceptor. In slices of tissue, 

 and presumably also in the intact tissue, the reaction is essentially 



