Glycolysis and the Pasteur Effect 207 



of enzyme activities in the extract showed some features of 

 interest. Hexokinase and phosphofructokinase, the adenosine 

 triphosphate (ATP)-utihzing enzymes of glycolysis, had the 

 lowest capacity. 3-Phosphogiycerate (PGA) kinase and pyruv- 

 ate kinase, the ATP-forming enzymes, as well as lactic 

 dehydrogenase, had the highest capacities, between 10 and 

 40 times that of ATP-utilizing enzymes. It should be empha- 

 sized that the capacity of an enzyme, defined as its activity, 

 measured in the presence of excess substrate and coenzymes, 

 is not representative of its role in overall glycolysis, but 

 conveys information with respect to its potentialities in 

 competitive systems. For instance, it was found that a 

 Pasteur-like effect can be produced by the addition of excess 

 mitochondria to a reconstructed system of glycolysis. When 

 the distribution of glycolytic enzymes was similar to that 

 found in extracts of ascites cells, namely, with PGA kinase 

 and pyruvate kinase present in high concentrations, a pro- 

 nounced inhibition of respiration (Crabtree-like effect) could 

 be obtained. With limiting amounts of these adenosine 

 diphosphate (ADP) transphosphorylating enzymes, no Crab- 

 tree-like effect was observed although glycolysis was proceed- 

 ing at an appreciable rate. This can be readily understood 

 since the experiments were so designed that the regeneration 

 of ADP from ATP was the rate-limiting step for both respira- 

 tion and glycolysis. In order to compete with the efficient 

 phosphorylation of ADP due to oxidative phosphorylation, 

 relatively large amounts of transphosphorylating enzymes 

 were required in the reconstructed system. 



The next step was to measure overall glycolysis in crude 

 extracts and homogenates of ascites cells. They were fortified 

 with cofactors and inorganic phosphate in order to study the 

 rate-limiting enzyme. It was found that, depending on ex- 

 perimental conditions, there could be 2 or 3 enzymes limiting 

 the rate of glycolysis. The limiting factors were determined 

 by adding individual, highly purified enzymes of glycolysis to 

 the crude extract. Their effect on lactic acid production is 

 shown in Table I. It can be seen that in dilute extracts (1 mg. 



