288 LIGHT AND LIFE 



terest that the ATPase and ADPase activities are also dependent npon 

 the same metal ions for activity in this enzyme preparation. 



The kinetic data, as well as differences in metal activator require- 

 ments for the active nucleotides, lend strong support to data pre- 

 sented earlier in the text which suggested that ATP functions in- 

 directly in this system by acting as a generating system for ADP and 

 AMP via an ATPase reaction. That this is, in fact, the case is 

 clearly shown by experiments using partially purified fractions of 

 Renilla luciferase, the results of which are presented in the following 

 sections. 



Preparation of DEAE-cellulose Fractions 

 The 0-75% ammonium sulfate fraction of the crude extract was 

 dialyzed against 0.002 M potassium phosphate buffer, pH 7.5, con- 

 taining 0.001 M GSH, for four hours at 0-3 °C. The dialyzed frac- 

 tion was then absorbed on a NaOH-charged DEAE-cellulose column 

 and eluted off the column with 0.05 M potassium phosphate, pH 

 7.5. The resulting column fractions showed a to 5-fold purification 

 based on specific activity measurements using ADP in the assay 

 system. 



Properties of DEAE-fractionated Luciferase 



Phosphatase Contetit and Nucleotide Requirement 



The data in Table 5 show that the phosphatase activity for both 

 ATP and ADP decreases approximately 10-fold in the DEAE frac- 

 tions as compared to tlie crude extract. Furthermore, when phos- 

 phatase activity is lost, the ability to produce light with ATP is 



TABLE 5 

 Phosphatase and Nucleotide-Stimulated Luminescence Activities of 

 Crude and DEAE-kractionated Renilla Luciferase 



Phosphatase Activity Luminescence 



Enzyme (Mmoles Pi/20 min) (Relative Lisjht Intensity) 



Added ATP ADP None AMP ADP ATP 



Conditions: Same as described under assay system except that the enzyme added in 

 a'l cases represents 0.25 mp; protein. When ATP was used for tiie luminescence assay, 

 5 Mmoles of MgCl. were added. MgCl2 was also incorporated into the phosphatase 

 assay at equimolar concentrations with that of the nucleotides. 



