852 LIGHT AND LIFE 



There is first a rajjid decrease (half-value time, 0.25 sec.) due to the 

 initial inhibition of the enzyme through accumulation of E-L-AMP. 

 This is followed by a slower rate of decay (half-value time, 13 

 seconds) governed in part by the production of pyrophosphate, which 

 tends to break up the inhibitory enzyme-oxyluciferin complex and 

 so to set free the enzyme once more. The latter effect is demonstrated 

 by the action of pyrophosphatase, which reduces the emission of light 

 in the system to a very low level that may be raised again through 

 the addition of pyrophosphate. Yet pyrophosphate added to the sys- 

 tem before the addition of ATP has an opposite effect, tending to 

 prevent the formation of the active luciferase-luciferin-AMP complex. 

 The authors believe that the firefly controls its flash by using this 

 pyrophosphate mechanism. 



LH2-AAIP, formed chemically by condensation of its two parts, 

 yields, as expected, an active substrate lor the luminous reaction in 

 the presence of luciferase but without ATP or Mg+ + . Only free 

 oxygen is needed. Free oxyluciferin (L) does not inhibit this re- 

 action, but the product inhibition (i.e., by E-L-AMP) is even more 

 striking than in the natural reaction involving luciferin, luciferase, 

 and ATP. Instead of a continuous production of light, the available 

 enzyme is rapidly bound and the emission of light ceases, although 

 it can be reinitiated by adding more enzyme. The contrast between 

 the two reactions (starting with LH^ or with LH^-AMP) suggests 

 that in the natural system ATP (or luciferin, or both) is capable to 

 some degree of freeing the enzyme from the inhibitory product of 

 the reaction. When the substrate, LH^-AMP, is added to enzyme, 

 pyrophosphate, if present, will compete with oxygen and reduce 

 the emission of light by increasing the reverse separation of the E-LH^- 

 AMP complex into enzyme, luciferin, and ATP. Magnesium ions of 

 course greatly enhance this effect of j^yrophosphate, and virtually 

 block the emission of light completely. Further demonstration of the 

 correctness of the scheme has been shown by incorjjorating P-^--labeled 

 pyrophosphate into ATP through the reaction just described; and by 

 inhibiting luciferase through the addition of chemically synthesized 

 LAMP to the luminous reaction system. 



When oxyluciferin is added to enzyme and ATP, there is a very 

 great decrease in its fluorescence at 514 ni/^. This property has been 

 useful in the study of a hydrolytic enzyme found in the firefly whidi 

 will hydrolyze both LH.rAMP and L-AMP when they are free, but 

 not when they are bound to luciferase. The fluorescence serves as an 

 intlicator of free oxvlucilerin. It is also useful in studying another 



