Discussion 89 



this DPN again (Hunter, F. E., Jr., Shutz, B., Malison, R., and Atchison, 

 A. (1958). Fed. Proc, 17, 247). Do you think there is any association 

 between this discharge phenomenon and the conversion of DPN^^I to 

 DPN? 



Slater: I am not famihar with this work of Hunter which you mention. 

 We have not determined whether some of the DPN or DPNH has gone 

 outside the mitochondria or whether it all remains inside. We shall 

 certainly test this. 



Backer: Prof. Lehninger, do these different DPN's react differently 

 with various dehydrogenases? 



Lehninger: There certainly is a great difference in reactivity toward 

 [3 -hydroxy butyric acid and malic dehydrogenases. In the digitonin 

 particles there is less than one mole of bound DPN per mole of cyto- 

 chrome a and this one is apparently concerned in phosphorylation. The 

 intact mitochondria have something like 40 moles of DPN. It is possible 

 therefore that you cannot see that one DPN molecule very well in a pool 

 of 40 when you are studying intact mitochondria spectrophotometric- 

 ally; the "pools" of DPN may have different functions. 



Slater : In our experiment, what we obtain is the appearance of extra 

 DPN, more than we have at zero time. We get this with ADP, inorganic 

 phosphate or DNP, but not with ATP. That is what links it with oxida- 

 tive phosphorylation. We were not looking for a DPN intermediate of 

 oxidative phosphorylation. It was found accidentally while Dr. Purvis 

 was trying to deplete mitochondria of DPN for his work on pyi'idine 

 nucleotide transhydrogenase. 



Holton : In reference to one of your mechanistic slides* with DPNH, 

 the total mechanism is very complex. Does your evidence for DPN '--'I 

 mean that one of these intermediate steps can be struck out from your 

 mechanism? For instance, the action of DNP could well be in breaking 

 this DPN ^1^ down. You have introduced another energy-rich inhibited 

 compound. Is there a possibility of simplification by cutting out one 

 step? 



Slater : The reason for introducing this extra step was that the ATPase 

 reaction, which involves this X'--'I, was not affected by the state of 

 oxidation and reduction of the respiratory chain. We had to take com- 

 ponents of the respiratory chain out of the ATPase reaction, and our 

 reasons for doing that still remain. Prof. Chance introduced the X for 

 a kinetic reason (Chance, B., and Williams, G. R. (1956). Advanc. 

 EnzymoL, 17, 65). Certainly, we would still retain the necessity for the X. 



Holton: We have recently come upon some independent evidence 

 that the compound whose breakdown is catalysed by DNP is formed 

 by a sequence of at least two reactions. Dr. Beechey, in our laboratory, 

 has been studying heart sarcosomes possessing respiratory control 

 (Beechey, R. B. (1959). Biochim. biophys. Acta, in press) using the 

 polarometric method of Chance and Williams (1955, Nature (Lond.), 175, 

 1120). He has found that their respiration attains approximately the 

 same rate with ADP as with optimal concentrations of DNP or dicouma- 

 rol; this suggests that the same reaction is limiting the rate with the 

 * [Not submitted for publication. — eds.] 



