276 LIGHT AND LllE 



This extract was heated at 100°C for IV2 minutes, cooled to room 

 temperature, and the denatured protein then removed by centrifuga- 

 tion. The supernatant solution contained Renilla luciferin, which 

 can be stored at -15°C lor weeks without significant loss of activity. 



Assay System 



Unless otherwise specified, the luminescence assay system consisted 

 of the following additions: potassium jDhosphate buffer, pH 7.5 (50 

 /;imoles) ; GSH (2 /^moles) ; ATP, ADP, or AMP (5 ^.imoles) ; Renilla 

 luciferin (0.2 ml) ; enzyme (0.5 mg protein) ; and water to 1.5 ml. 

 In the experiments reported, ADP was used in preference to ATP, 

 since the latter compound functions indirectly in this system. All re- 

 actions were carried out at 30 °C unless otherwise stated. 



Properties and Characteristics of the System 



Requirement for Adenine-Containing Nucleotides 



Crude extracts from R. reniformis give a relatively weak lumines- 

 cence that is stimulated approximately 10- to 30-fold (depending 

 upon the extract) upon addition of ATP, ADP, or AMP. These 

 results are presented in Table 1. Enzyme preparations can be ob- 

 tained, by repeated precipitations with saturated ammonium sulfate, 

 that are stimulated 100-fold upon the addition of adenine-containing 

 nucleotides. Adenosine is inactive in this system, as well as the mono- 



TABLE 1 

 Effect of Nucleotides on the Luminescence of R. reniformis Extracts 



Experiment Addition Relative Light Intensity 



I None 1 6 



ATP 445 



ADP 390 



AMP 440 



Adenosine 16 



GTP, CTP, or UTP 16 ± 1 



GMP, CMP, or UMP 16 ± 1 



II None 20 



ADP 450 



GDP 23 



GDP 36 



UDP 122 



Conditions: Same as described under assay system. 5 ;umoies of each nucleotide or 

 nucleoside were added as indicated. ATP, ADP, AMP, adenosine triphosphate, 

 diphosphate, and monophosphate, respectively. GTP, CTP, UTP, guanosinc, cytidine, 

 and uridine li iphosphales. (.Ml'. CMP. I 'Ml', guanosine, cytidine, and uridine 

 monophosphates. (.1)!', CDl', 11)1', guanosinc, (Aiidiiic, and uridine diphosphates. 



