170 T. E. CONOVER 



and with added glucose-6-phosphate and TPN, very little consumption 

 of oxygen was observed, as is shown in Fig. i. This is due to the fact 

 that, as has been reported previously by Pullman and Racker [16], TPNH, 

 which is formed from the oxidation of glucose-6-phosphate, is not readily 

 oxidized by mitochondria. If vitamin K3 in low concentration was added, 

 however, there was initiated a rapid oxygen uptake, indicating a rapid 



10 15 

 minutes 



20 



Fig. I. Oxidation of glucose-6-phosphate by liver mitochondria in the 

 presence of soluble cytoplasm and vitamin K3. The complete system contained 

 per Warburg vessel i -o /^tmoles TPN, 20 jumoles glucose-6-phosphate, 50 jumoles 

 tris buffer (pH 7-4), 30 /^moles orthopbosphate (pH 7 "4), 10 /imoles MgCl,, 

 5 /tmoles adenosine triphosphate, 60 ;u,moles glucose, an excess of yeast hexokinase, 

 225 /xmoles sucrose, 0-03 /tmole vitamin K^, dialyzed supernatant fluid centri- 

 fuged at 105 000 X g from 450 mg. rat liver, and mitochondria from 500 mg. rat 

 liver. Final volume, 3-0 ml. Temperature, 30 . Reading begun after 5 min. 

 thermoequilibration. 



1 . Complete system. 



2. No vitamin Kg. 



3. No mitochondria. 



oxidation by the mitochondria of the TPNH formed in the incubation 

 medium. 



This respiration was insensitive to Amytal but was inhibited by Anti- 

 mycin A and cyanide (Table I). Most importantly, it was observed to be 

 sensitive to dicoumarol at concentrations of io~^ M or less, which is a very 

 characteristic property of DT diaphorase [9]. 



The implication that DT diaphorase was involved in the oxidation of 

 extramitochondrial TPNH in this system was supported by the duplication 

 of this system with isolated enzymes. Mitochondria freshly prepared from 



