244 



Van R. Potter and Hermann Niemeyer 



of TPNH obtained by incubating it in a boiling bath for a few 

 minutes strongly inhibited glucose uptake, lactic acid produc- 

 tion and phosphate disappearance (Fig. 9). 



Nevertheless, one preparation of TPNH that had the 

 spectrum corresponding to a very pure compound and that 

 was further purified of possible metal contaminants by passing 



•A GLUCOSE I +A LACTATE 



o-o CONTROL D-ciTPN 



A ORTHOPHOS- 

 .^TPNH PHATE 



-I 



50 100 50 100 50 100 



AGED LIVER MITOCHONDRIA (mg-equiv.) 

 Fig. 10. Effect of TPN and TPNH on glycolysis. Influence of 

 mitochondria. Each flask contained in a 1 -S-ml. volume, 0-5 ml. 

 brain supernatant, variable amounts of mitochondria, 7-5 [lm 

 glucose, 3 [iM ATP, 3 \im ADP, • 5 [jlm DPN, • 1 (jim cytochrome c, 

 6 (xM-MgCla, 10 [JLM potassium phosphate pH 7-45, 35 [jlm Tris 

 buffer pH 7-45. 3 jzm TPN or TPNH when indicated. 15 m-KCI 

 to maintain isotonicity. 



it through a Dowex-50 column, exhibited an effect on glyco- 

 lysis very similar to that of purified TPN (Fig. 10). 



TPN -lactic dehydrogenase — To understand the mechanism of 

 action of TPNH it was important to learn that brain super- 

 natant has a TPN-dependent lactic dehydrogenase activity, 

 similar to that found in rat liver (Navazio, Ernster and 

 Ernster, 1957). This enzyme can bring about the oxidation of 

 TPNH in the presence of pyruvate. It is not known if this is a 

 separate enzyme from the DPN-lactic dehydrogenase. The 



