176 T. E. CONOVER 



which were preincubated with iodoacetate in order to deplete the level of 

 lactate and pyruvate and therefore lower the transhydrogenase activity. It 

 can be seen in Fig. 6 that this preincubation has not changed greatly the 

 pattern of citrate oxidation. Vitamin K3 still stimulated respiration to 

 some extent. Most striking, however, is that the citrate respiration was 

 now markedly inhibited by dicoumarol. 



It would therefore appear that the DT diaphorase system can be demon- 

 strated in the intact cell with liver slices in the presence of added vitamin 

 K3 under conditions where the transhydrogenase activity of the cytoplasm 

 is low. It is difficult, however, to ascertain from this whether DT diaphorase 

 is able to function in the respiration and oxidation of reduced pyridine 

 nucleotide in normal conditions. Under usual slice conditions, particularly 

 in the presence of Amytal where lactate levels are high, the activity of 

 DT diaphorase may be masked by the transhydrogenase activity. 



D Without dicoumarol 

 Q With dicoumarol 



4- 



3- 



o 2 



I - 



citrate citrote Citrate 



Vit Kj-bis Vit Kj-bis Vit Kj-bis. 



TPN 



Fig. 6. The dicoumarol sensitivity of the Amytal-resistant respiration of 

 iodoacetate-pretreated liver slices. Conditions as in Fig. 4 except that the slices 

 were preincubated for 20 min. at 37 -5 in the presence of 2 x lo"'* m iodoacetate. 

 Time measured, 60 min. [13]. 



Several of the properties of DT diaphorase would suggest some 

 limitations in its function in the cell. From a Lineweaver-Burk plot for 

 the enzyme it was seen that the Michaelis constants for the enzyme are 

 high; o-i8 mM for DPNH and 0-13 mM for TPNH. Albumin, which 

 activates the enzyme in the purified state, gives a marked lowering of the 

 Kj^j to o-o8 mM and 0-04 mM respectively. TPNH in both cases has a 

 somewhat lower K^^/ than has DPNH. It would seem that though the 



