146 



HARLYN HALVORSON 



Minutes 



Fig. 3. Cofactor requirements for glucose oxidation by spore extracts. 

 See text for preparation of extract. Extract was dialyzed overnight against 

 water at 5C. The Warburg flask contents: sidearm. 1 mg of glucose, and 

 SxlO-"^ M DPN, TPN. or 0.2 ml of 0.05 M ATP where indicated; center well. 

 0.2 ml 30 per cent KOH; main compartment, 1 ml of enzyme preparation, 

 10 /^mole inorganic phosphate, spore ash and vitamins where indicated. Total 

 volume 2.1 ml. Reaction was run at 30C under atmospheric oxygen. 



5C. for 30 minutes and the debris removed by centrifugation at 10,000 

 limes gravity for 20 minutes. The supernate so obtained was free of spores. 

 Approximately .50 per cent of the spores were ruptured by this treatment. 



The cofactors required for optimal glucose oxidation by crude spore ex- 

 tracts are shown in Fig. 3. Optimal activity was observed in the presence 

 of yeast extract. This requirement could be fully replaced by diphosphopy- 

 ridine nucleotide (DPN) and partially by triphosphopyridine nucleotide 

 (TPN). However, when the extracts were dialyzed, only 65 per cent of the 

 activity was obtained in the presence of DPN. In these dialyzed prepara- 

 tions a partial requirement for both ATP and inorganic phosphate was 

 observed. 



The oxidative capacities of crude spore extracts supplemented with TPN 

 or DPN are shown in Fig. 4. Glucose and pyruvate are actively oxidized 

 in the presence of DPN. Gluconate and 2KG oxidations are TPN depend- 

 ent. On the other hand, members of the Embden-Meyerhoff glycolytic sys- 

 tem (G-6-P, F-6-P, F and HDP), as well as arabinose and ribose, are in- 

 variably inactive. 



A kinetic analysis of the glucose oxidation by crude spore extracts ia 

 shown in Fig. 5. It is clear that an active system is present capable of con- 

 verting glucose to pyruvate. The low recoveries of pyruvate are partially 



