MILTON J. CORMIER 



291 



10 



15 



20 25 



TIME (seconds) 

 Fig. 10. Kinetics of the reaction upon addition of preformed intermediate. 



Requirements for Formation of the Intermediate 



A study of the requirements for the formation of the interinediate 

 was possible using a DEAE-fractionated enzyme of the type used to 

 obtain the data in Table 5. This enzyme preparation contains a 

 negligible amount of adenine-containing nucleotides, on the basis 

 of luminescence determinations, and is essentially devoid of luciferin. 

 As seen from Table 7, the formation of the intermediate requires the 

 presence of both ADP and luciferin. The relatively small light signal 

 obtained when the complete system minus ADP is used is presumably 

 due to low levels of contaminating nucleotides in the crude luciferin. 

 Although the data are not shown, AMP is about equally active under 

 these conditions, whereas ATP plus magnesium ion is totally inactive. 



In addition to ADP and luciferin, the enzyme preparation is also 

 required for intermediate formation. The intermediate is not formed, 

 for example, if ADP and luciferin are preincubated under anaerobic 

 conditions in the absence of enzyme. 



The above data suggest that a heat-stable intermediate is formed 

 by an enzyme-catalyzed reaction involving luciferin and either ADP 



TABLE 7 

 Requirements for Intermediate Formation 



Test System 



Relative Light Intensity 



Complete 



' minus luciferin 



minus ADP 

 " minus ADP and luciferin 



" minus enzyme 



162 



6 



36 



3 



8 



