144 LARS ERNSTER 



kindly placed at our disposal by Dr. M. Klingenberg in Marburg. As shown 

 in Fig. I, the restoration ot respiration by vitamin K3 in the amytal- 

 blocked system was accompanied by an abrupt increase of the light 

 absorption difference at 434 490 m/x, indicative of a reduction of cyto- 

 chrome h. 



SEPARATION OF DT DIAPHORASE AND DPNH OXIDASE 



In order further to fortify the concept that DT diaphorase does not 

 participate in the main DPNH oxidase pathway, an attempt was made to 

 separate the two systems. This proved possible by exposing liver mito- 

 chondria to disruption by a rapidly rotating Super-Thurrax blendor, 

 followed by differential centrifugation, essentially according to the 

 procedure employed by Kielley and Kielley [24] in their studies of mito- 

 chondrial ATPase. The procedure results in three submitochondrial 

 fractions: a soluble fraction, a light pellet, and a heavy pellet. 



D DPNH 

 □ TPNH 



.£ 2 



.9 Light Heavy >- 



-D Sup. pel. pel. % 



I ^ V ' o 



o Submitochondrial fractions <^ 



Fig. 2. Diaphorase activities of submitochondrial fractions prepared according 

 to Kielley and Kielley (1953) (from Danielson, Ernster, and Ljunggren [25]). 



In examining the DPNH and TPNH diaphorase activities of these 

 fractions it was found (Fig. 2) that the soluble fraction contained virtually 

 the entire TPNH diaphorase activity of the original mitochondria, accom- 

 panied by an equal DPNH diaphorase activity, whereas the two pellets 

 exhibited only DPNH diaphorase activities. Moreover, the activities found 

 in the soluble fraction were markedly activated by Tween and strongly 

 inhibited by dicoumarol (both of these properties are characteristic of 

 DT diaphorase), whereas the activities of the pellets were not influenced by 

 these agents (Table IV). The selectivity of the inhibition by dicoumarol 

 between soluble and pellet DPNH diaphorase activities is illustrated in 

 Fig. 3. This treatment of mitochondria thus resulted in a selective solubil- 

 ization of DT diaphorase. 



