228 



LIGHT AND LIFE 



7 suggests that the latter mechanism is more nearly correct. The re- 

 sults in the figure indicate that in a mixture of luciferase and L-AMP 

 in which the latter is at a concentration greater than that of the 

 enzyme there is a fluorescence increase upon the addition of ATP. 

 The fluorescence increase corresponds to the hydrolysis of the amount 

 of L-AMP which is in excess of luciferase. A similar experiment 

 carried out with LAMP concentration less than the enzyme indicates 

 very little effect of ATP on fluorescence. These results can be in- 

 terpreted most readily in terms of the second mechanism described 

 above and on the basis of the high affinity of the L-AMP for the 

 luciferase. If ATP in some way causes the labilization of the luciferase- 

 bound L-AMP, with subsequent hydrolysis, this would leave free 

 oxyluciferin on the enzyme surface. If there is no free LAMP in 

 solution, the oxyluciferin molecule would react immediately with 

 ATP to produce another molecule of E-L-AMP and pyrophosphate. 

 However, this w^oidd not always be the case when free LAMP is 

 present. Because of the extremely high affinity of L-AMP for luci- 

 ferase, L-AMP could readily replace oxyluciferin on the enzyme sur- 

 face and subsequently be hydrolyzed. The other observation in sup- 



80 



(L-AMP) < (E) 



12 



6 8 



MINUTES 

 l"ig. 7. i:(rcct of A IP oil tlic liydiolysis of L -AMI'. Miiorcscciice increase is due 

 to the hydrolysis of L-.AMP to L and AMP. Excitation at 330 mfi; emission 

 measured at 540 m/x. See text. 



