602 LIGHT AND LIFE 



plasts, and some properties and kinetics of the reaction under a 

 variety of conditions. Of interest are certain experiments in which the 

 ATP level is lowered by illumination. The stimulation of photo- 

 phosphorylation by added folic acid is also reported. 



Material and Methods 



(a) Chloroplasts. Washed spinach chloroplasts were prepared as 

 described elsewhere (11) and were stored at 0°C in the dark until 

 use. 



(b) Firefly enzyme. Crude firefly luciferase (26) was freshly pre- 

 pared by exhaustively grinding 12 vacuum-dried firefly lanterns 



iPhotirius pyralis) with sea sand in a mortar and pestle, followed by 

 extraction with 7 ml of 0.01 M phosphate buffer, pH 7.4 at 0°C. 

 The solution was filtered into a test tube and stored until use at 

 0°C. 



(c) The chloroplast and firefly enzyme mixtures were illuminated 

 with a Bausch &: Lomb 6 v-18 amp ribbon filament microscope lamp 

 burned at an overvoltage. The beam was coUimated and projected 

 through a water-filled heat filter and four red filters in series. The 

 four red filters were found to be necessary to remove all traces of blue 

 light from the illuminating beam. The reaction mixture was illumi- 

 nated in a thin Incite cuvette. The blue-green fraction of light emitted 

 within the chamber because of the luminescence of the firefly enzyme 

 was transmitted directly to the end window of a 5819 RCA photo- 

 multiplier by two filters sandwiched between the cuvette and photo- 

 multiplier. 



The direct current output of the detector was amplified by an 

 Aminco photometer whose output was applied to a one-second Brown 

 Recorder. High voltage was furnished by a Baird Atomic Instrument, 

 Model 312. 



Details of the illuminating and measuring arrangement are illus- 

 trated in Fig. 1. 



(d) ATP, ADP, AMP, DPN, TPN, DPNH, TPNH, cytochrome r, 

 FMN, PMS (phenazine methosulfate) , and myokinase were obtained 

 from Sigma Chemical Company, St. Louis, Missouri. Folic acid, 

 menadione, and a-tocopherol were obtained from Nutritional Bio- 

 chcnii(al Corporation, Cleveland, Ohio. 



Experimental Results 



(a) Kinetics ol the reaction. In Fig. 2 is illustrated the response of 

 the system to successive periods of light and dark. Note particularly 



