BENTLEY GLASS 853 



reaction, which like one nientionecl prexiously indicates that ATP 

 can tree the enzyme ironi the inhibitory j^rockict LAMP. In a Hght- 

 proclucing reaction system containing kicilerin, ATP, luciferase, and 

 magnesiinii, pyrojjhosphate will continue to be produced long after 

 the product inhibition of luminescence has occurred, provided pyro- 

 phosphatase is present to pull the reaction. Pyrophosphate is still 

 formed when oxyluciierin replaces luciferin in the system. It seems 

 that ATP in some way hydrolyzes the oxyluciierin adenylate on the 

 surface of the enzyme, so that the oxyluciierin is free to react with 

 ATP to release pyrophosphate. 



The dissociation constant of the complex E-L-AMP has been de- 

 termined, and from the linearity of the dissociation curve at various 

 concentrations of LAMP and of enzyme, McElroy and Seliger con- 

 clude that the luciferase molecule has a single site for the luminotis 

 reaction and that a single molecule of L-AMP is bound at this site. 

 Further evidence for this relationship is seen in the observation that 

 even a thousand-fold excess of enzyme in relation to substrate does 

 not reduce the height of the initial flash below that expected on a 

 linear relation of flash height to enzyme-substrate ratio. 



Coenzyme A has an interesting stimulatory effect upon the lu- 

 minous reaction. In a system in which the luciferin concentration is 

 greater tlian that of the enzyme, and which has reached the stage 

 of slow decay in the emission of light, the addition of CoA stimulates 

 the production of light in direct proportion to concentration. It has 

 been shown to do this by relieving the product inhibition by react- 

 ing with the E-L-AMP to produce AMP, L-CoA, and free enzyme. 

 The reaction is reversible. Oxyluciferyl-CoA will react with cysteine 

 or glutathione. The stages in the reaction may be followed spectro- 

 photometrically, since the fltiorescence of L-CoA is only about 2% 

 of that of oxyluciierin, and that of oxyluciferyl-cysteine is about 50% 

 of that of oxyluciferin. 



The effects of several factors upon the luminous reaction have been 

 studied. In glycyl glycine buffer the pH optimum is at 7.8. In phos- 

 phate buffer the intensity of the light is greatly reduced. The tempera- 

 ture optimum of the reaction is 25° C. The activation energy is cal- 

 culated to be 18 kcal. Increase in the oxygen concentration enhances 

 the emission of light up to a concentration of 0.5%, w'hile lowering 

 the Oo concentration does not materially reduce the light intensity 

 until it falls below 0.1%. Under anaerobic conditions E-LHo-AMP 

 accumulates in the system, so that upon readmission of oxygen there 

 is a rapid reaction marked by a flash. The duration of this oxygen 



