220 E. C. SLATER, M. J. BAILIE AND J. BOUMAN 



It should be emphasized that all of these experiments were carried out in 

 the presence of nicotinamide, which prevents the destruction of DPN +. 

 In six of the eight experiments, we added dinitrophenol which, in agree- 

 ment with Chance's observations with other uncoupling agents, brought 

 about oxidation of much of the DPNH. There was an increase in the 

 (DPN + + DPNH) content of between 0-3 and 2-4 (average i • i) /xmoles/g. 

 protein. 



Thus, during the incubation with substrate a part of the total DPN 

 disappears into a compound which does not react in our enzymic method. 

 It reappears as DPN + on addition of dinitrophenol. This compound is not 

 TPN +. In three experiments, in which an average of i • i /^mole (DPN + + 

 DPNH)/g. protein disappeared during incubation, 0-25 /xmole (TPN+ + 

 TPNH)/g. protein also disappeared. In four experiments, 0-3 ju,mole 

 (TPN + + TPNH)/g. protein appeared after addition of the dinitrophenol. 



Recently, this experiment has been repeated with four preparations 

 using a spectrophotometric method. There is much less variation, which 

 may be partly due to the greater accuracy of the spectrophotometric assay, 

 but is also probably due to the fact that the experiments were carried out 

 in close succession with mitochondrial preparations which were probably 

 very similar to one another. Qualitatively, the same picture is shown (see 

 Fig. 2 (B)), but the amount of total pyridine nucleotide disappearing was 

 rather less — between 0-2 and 0-7 jumole/g. protein. 



Three experiments (fluorimetric) were also carried out with succinate 

 in place of glutamate. There was some loss of DPN +, while the DPNH 

 content did not increase (see column 4, Table VII). 



From these experiments, we can conclude that incubation with sub- 

 strate in the absence of inorganic phosphate causes some of the diphos- 

 phopyridine nucleotide to disappear, and that what disappears can be 

 largely recovered again by the addition of dinitrophenol. The amount of 

 DPN disappearing in this way was sometimes quite large, but was often 

 only small in experiments which appeared to be carried out in the same 

 way with identical material. We do not understand the reasons for these 

 differences and, at present, our preparations of rat-liver mitochondria are 

 discouraging material for the study of the Purvis compounds. For this 

 reason, we have recently turned to other mitochondria. 



However, before leaving the experiments summarized in Fig. 2 and 

 Table VII, it is worth while drawing attention to the state of oxidation of 

 the pyridine nucleotides in the controlled and active states. In these 

 experiments, respiration was controlled or inhibited by lack of inorganic 

 phosphate. In this controlled state, a substantial proportion of the diphos- 

 phopyridine nucleotide is in the oxidized form, nearly half in the first 

 series of experiments (Fig. 2 (A)). This is similar to Klingenberg and 

 Slenczka's finding when respiration was inhibited by lack of ADP. In fact. 



