MILTON J. CORMIER 277 



phosphates and tiipliosjihatcs of guanosine, cytidine, and uridine. 

 The diphosphates ol guanosine and cytidine are also inactive, al- 

 thonoh some activity was exhibited by uricHne dipliosphate. Whether 

 the hitter activity is due to contamination with adenine-containing 

 nucleotides is not certain, altliough on the basis of concentration 

 curves (see Fig. 1) , a 1 per cent contamination with any one, or a com- 

 bination, of the adenine-containing nucleotides would have been suffi- 

 cient to account for the light observed. Furthermore, since UTP and 

 UMP are inactive, it appears unlikely that uridine-containing nu- 

 cleotides are active in this system. Thus, luminescence in extracts 

 of R. reuiformis is dependent upon the presence of adenine-containing 

 nucleotides and is specific for the adenine moiety, at least in the 

 initial step of the reaction. 



Since light emission in the firefly requires a primary activation by 

 ATP (10), and since the Renilla system responds to either ATP, 

 ADP, or AMP, there appeared to be a good possibility that the two 

 systems were similar with respect to nucleotide requirements. The 

 purity of the ADP and AMP was therefore checked for contamina- 

 tion with ATP. The AMP used in these experiments did not contain 

 ATP as determined by ATP analysis using the firefly technique (13, 

 17) . The sensitive light-detecting apparatus used in these experi- 

 ments readily allowed the detection of 10-^ ^g ATP/ml. Likewise, 

 insufficient amounts of ATP were found in the ADP preparations 

 to account for the light observed. Furthermore, various commercial 

 sources of ADP and AMP were found to be equally effective. In 

 addition, the AMP used was not contaminated with ADP, which was 

 measured by coupling adenylate kinase to the firefly system. The 

 activities exhibited by AMP and ADP, therefore, were not due to con- 

 tamination w4th ATP. 



The possibility existed, however, that the AMP and ADP activities 

 could be attributed to phosphorylations in the crude extract leading 

 to the formation of ATP. This possibility can also be ruled out, 

 since AMP and ADP are fully effective in a phosphate-free system, 

 as well as in the presence of 2,4-dinitrophenol (10-^ M, final concen- 

 tration) . The method of Fiske and Subbarow (6) was used to de- 

 termine the absence of inorganic phosphate in these experiments. In 

 addition, adenylate kinase could not be demonstrated in the crude 

 extracts since, under anaerobic or aerobic conditions, no ATP for- 

 mation could be detected from ADP using the firefly assay. Thus, the 

 AMP and ADP activities are not due to contamination with ATP 

 or to their conversion to ATP. 



