ir. I). Md'JJiOY AND H. H. SULK. Eli 



223 



2 3 4 5 6 7 



TIME - MINUTES 

 fig. 3. EfTect of inorganic pyrophosphatase and pyrophosphate on light emission. 

 The initial rapid decline of light intensity after the addition of ATP is due to 

 product inliibition and the removal of pyrophosphate which favors the removal of 

 L-.AMP from the enzyme, as described in Fig. 1. The addition of pyrophosphate 

 at two minutes stimulates light emission until it is removed by hydrolysis. Inor- 

 ganic pyrophosphatase is present to hydrolyze the pyropliosphate produced in the 

 activation reaction. 



reaction, the light intensity curve seen in Fig. 2 is altered to give the 

 initial response indicated in Fig. 3. After the initial flash has decreased 

 to the low level luminescence a secondary addition of pyrophosphate 

 results in a stimulation of light emission. The luminescence remains 

 at this high level until all of the pyrophosphate has been hydrolyzed. 

 Ions such as fluoride, which are known to inhibit pyrophosphatase 

 activity, greatly influence the response to added pyrophosphate. 



It is significant that pyrophosphate, even in low concentration, is a 

 jjotent inhibitor of the kmiinescent reaction if added prior to the 

 ATP. Apparently it can compete with the latter to form an inactive 

 intermediate with the luciferin-luciferase system. A large amount of 

 data, some of which will be presented later, indicates quite clearly 

 that the oxidized luciferyl-adenylic acid derivative is tightly com- 

 plexed to the enzyme and effectively removes the latter from catalytic 

 activity. Low concentrations of pyrophosphate react with the enzyme- 



