142 LARS ERNSTER 



was blocked by the addition of i mM amytal, it could be completely 

 restored by vitamin K3 and was then strongly inhibited by dicoumarol. 

 The vitamin K3-induced respiration was, in accordance with the observa- 

 tions of Colpa-Boonstra and Slater [23], sensitive to antimycin A. The 

 antimycin A inhibition could be overcome to some extent by adding 

 cytochrome c (not shown). 



From these findings it was concluded that in intact liver mitochondria 

 there exist two pathways of antimycin A-sensitive DPNH oxidase, of 

 which only one, characterized by a sensitivity to amytal, functions under 



TABLE II 



Effect of Vitamin K., and Various Inhibitors on the Respiration of 

 Mitochondria in the Presence of Amytal 



(from Conover and Ernster [22]) 



Additions /xatoms oxygen 



None 5 • 49 



Vitamin K3 5 57 



Dicoumarol 5 32 



Amytal 0-22 



Amytal + vitamin K3 5 '25 



Amytal + vitamin K3 + antimycin A 1-57 



Amytal + vitamin K3 + KCN 118 



Amytal + vitamin K3 + dicoumarol i ■ 1 3 



The complete system contained per Warburg vessel: 10 /unioles glutamate, 

 20 fimoles tris buffer (pH 7 '4), 20 //.moles orthophosphate (pH 7 "4), 4 /xmoles 

 MgClo, 2 /xmoles adenosine triphosphate, 24 /xmoles glucose, an excess of yeast 

 hexokinase, 50 /itmoles sucrose, and mitochondria from 200 mg. rat liver. The 

 amounts of the additions were as follows: 5 x iq-^ /xmole vitamin K3, lO"* /itmole 

 dicoumarol, 10 /tmole amytal, i [xg. antimycin A, and i-o /xmole KCN. Final 

 volume, I o ml. Temperature 3o\ Reading begun after 6 min. thermoequilibra- 

 tion. Time measured, 20 min. 



normal conditions. Another pathway, characterized by a sensitivity to 

 dicoumarol and proceeding probably via DT diaphorase, is not functioning 

 normally in the terminal oxidation of DPNH, because it lacks a link to the 

 cytochrome system, but it can be brought into reaction by adding an 

 artificial link such as vitamin K.5. 



The phosphorylation arising from the vitamin Kg-induced respiration 

 in the amytal-blocked system yielded a P/0 ratio that was about one unit 

 lower than that of the normal system (Table III). However, the P/0 ratio 

 obtained with succinate as substrate also was lowered under these condi- 

 tions. In other words, it cannot be decided with the evidence presently 

 available, whether or not the vitamin Kg-induced by-pass of the amytal- 

 sensitive site involves the loss of a phosphorylation. 



