1 72 T. E. CONOVER 



rat liver were incubated in an isotonic buffered medium containing TPN, 

 glucose-6-phosphate, purified Zwischenferment, phosphate, Mg + +, ATP, 

 hexokinase, and glucose. As is shown in Fig. 2 the extramitochondrially- 

 generated TPNH was not oxidized under these conditions to any ap- 

 preciable extent by the mitochondria in agreement with the previous 

 experiment. Addition of vitamin K3 produced a two- to threefold stimula- 

 tion of the total respiration. Addition of purified cytoplasmic DT dia- 

 phorase [9] to the vitamin Kg-stimulated system gave a further two- to 

 threefold stimulation of the respiration. This rate of respiration was close 

 to that obtained maximally with succinate or glutamate as substrate and 

 it may represent the limit of the cytochrome system to react with oxygen 

 rather than that of TPNH to react with the cytochrome chain. 



TABLE II 

 Effect of Some Inhibitoks on the Mitochondrial Oxidation of Extra- 



MITOCHONDRIAL TPNH MEDIATED BY DT DiAPHORASE AND VlTAMIN K, 



Additions ( + ) or omissions ( — ) Relative oxygen consumption 



Complete system 100 



+ 2 X 10^ M Amytal 118 



+ I /xg. Antimycin A 20 



+ lO"^ M cyanide 24 



+ lO""® M dicoumarol 24 



— DT diaphorase, — vitamin K;, 20 



Conditions as in Eig. 2. Respiration of complete system was 5-56 /diatoms 

 oxygen /20 min [3]. 



The pattern of inhibition by various inhibitors was identical in this 

 system to that in the previous one (Table II). The inhibition of the 

 respiration by Antimycin A and cyanide is in agreement with the report of 

 Colpa-Boonstra and Slater [2] on the oxidation of reduced vitamin Kg by 

 mitochondria. This inhibition by Antimycin A suggests the entrance of 

 electrons into the respiratory chain at, or above, the site of Antimycin A 

 inhibition, probably at the level of cytochrome h. This is supported by 

 other experiments which are reported elsewhere [8]. 



As DT diaphorase can reduce a wide variety of quinones tested [8, 9] 

 it was assumed that this stimulation of the oxidation of TPNH was a 

 general property of such quinones. However, as is shown in Table HI, it 

 was highly specific for 2-methyl-naphthoquinones, and in particular, for 

 vitamin K3. Such a specificity increased the anticipation of a biological 

 role for such a system. While it was recognized that vitamin K;5 is a highly 

 artificial material for a biological system it was felt that possibly some 



