MILTON J. CORMIER 289 



also lost. Oil the other hand, a loss of phosphatase activity has 

 little or no effect on the ability to stimnlate light production by 

 ADP or AMP. The stimulation of limiinescence by ATP can be 

 restored in the DEAE fractions by the addition of commercial prepara- 

 tions of alkaline phosphatase. These results are presented in Table 6. 

 Thus, the activity observed with ATP in the crude extracts can be 

 attributed to phos])hatase action which results in the generation of 

 ADP and AMP from ATP. The functional nucleotide, therefore, 

 in the luminescence of the sea pansy is either ADP or AMP, or both. 

 It has not been possible, on the basis of kinetic data, and using 

 partially purified preparations, to distinguish between the activities 

 of the latter two nucleotides. 



TABLE 6 



Effect of Alkaline Phosphatase on the ATP Response in 



DEAE Fractions 



System Relative Light Intensity 



Complete 234 



minus ATP 16 



" minus phosphatase 26 



" minus phosphatase (ADP replacing ATP) 224 



" (ADP replacing ATP) 211 



Conditions : Same as under assay system except that 1 mg of crude alkaline phos- 

 phatase and 5 /xmoles of MgClj were added. 



Mechanism Studies 



Evidence for Formation of an Intermediate 



If crude Ren ilia luciferase is added to a standard assay system under 

 anaerobic conditions, and preincubated under anaerobic conditions 

 for varying lengths of time prior to flushing with oxygen, one obtains 

 kinetic data of the type illustrated in Fig. 9. The system is flushed 

 with oxygen for 20 seconds beginning at zero time (after 20 minutes 

 anaerobic preincubation) in the case of Figure 9A, and after 40 

 minutes anaerobic preincubation in the case of Figure 9B. Absolutely 

 no light can be detected unless oxygen is introduced. The fact that 

 the time required to reach half the steady-state value is approximately 

 2 seconds as compared to 3.3 minutes for ADP kinetics (see Fig. 8) 

 suggests the formation of an intermediate during anaerobic preincu- 

 bation. Although the decay of limiinescence from the steady state 

 is relatively rapid, as shown in Figure 9B, 10 to 15 minutes is re- 

 quired to return to a level near the normal steady-state value where 



