11. 1). Mcelroy and h. h. seliger 227 



in ihe presence of ATP ami hicifeiin. It is of interest in this con- 

 nection (hat the dieniically synthesized inosinic acid derivative of 

 oxyUicifcrin, LIMP, is also an extremely i)otent inhibitor, while 

 the IMP derivative of LH^ is not effective for light production (21) . 



Fluorescence of Oxyliiclfeyin and Oxyhicijeryl-Adeuylalc 



One of the interesting changes in the properties of oxyluciferin in 

 the enzymatic reaction Avith ATP is the tremendous decrease in the 

 intensity of fluorescence. Normally the fluorescence of oxyluciferin 

 gives a strong emission peak at 514 millimicrons (Fig. 6) . However, 

 when ATP and enzyme are added to such a reaction mixture the 

 fluorescence intensity of E-L-AMP at this wave length decreases dra- 

 matically. Other fluorescence properties of L-AMP will be discussed 

 later. Knowing these fluorescence properties of L-AMP, it has been 

 possible to study a number of interesting reactions of the enzyme- 

 L-AMP complex (1). For example, an enzyme has been found in the 

 firefly Avhich wiW catalyze the hydrolysis of both LAMP and LHo- 

 AMP. We have followed the hydrolysis of the L-AMP by observing 

 the increase in the fluorescence upon the formation of the free oxy- 

 luciferin. This hydrolytic enzyme will not attack L-AMP when it is 

 tightly bound to the luciferase. However, when the hydrolase is 

 present in the solution of luciferase-L-AMP there is a very slow 

 but definite recovery of enzymatic activity. This can be explained 

 in terms of the equilibrium between the enzyme-L-AMP complex 

 and free enzyme and free L-AMP. 



Another interesting aspect of the enzyme-L-AMP complex has been 

 studied by using fluorescence as an indicator of free oxyluciferin. 

 A light-producing reaction mixture containing luciferin, ATP, luci- 

 ferase, and magnesiimi continues to produce pyrophosphate long 

 after the enzyme is inhibited for light production, provided pyro- 

 phosphatase is present to pull the reaction. The same property of 

 the system is observed if luciferin is replaced by oxyluciferin (21) . 

 The only reasonable conclusion, therefore, is that the enzyme-L-AMP 

 complex is capable of causing the hydrolysis of ATP to adenylic acid 

 and pyrojihosphate. There are several possible mechanisms for the 

 interaction between ATP and the E-L-AMP complex. Luciferase-L- 

 AMP could act as a unit to catalyze the hydrolysis of ATP. If this 

 were true, the AMP moiety of LAMP would not be replaced by 

 ATP. Another jjossibility is that ATP in some way causes the hy- 

 drolysis of luciferase-boimd L-AMP, after which oxyluciferin reacts 

 with ATP to release pyrophosphate. The evidence presented in Fig. 



