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Light-Dependent Reductions in a Cell- Free System* 



WOLF VISHNIAC, Department of Microbiology, Yale University, New 



Haven, Connecticut 



A few years ago I reported on a cell-free system obtained from 

 spinach leaves which could be shown to require chlorophyll for a light- 

 dependent reduction of TPN. To recapitulate briefly, green granules 

 were obtained from macerated leaves and washed repeatedly by 

 centrifugation. The particles were then extracted with acetone at 

 — 5°C., on occasions also with butanol, until the washings were color- 

 less. An aqueous extract of the resultant greenish powder yielded a 

 colorless or yellowish solution; 2.0 ml. of such an extract in phosphate 

 buffer at pH 7.0, mixed with 0.05 ml. of an ethanolic extract of lyo- 

 philyzed chloroplasts and incubated anaerobically in the light, re- 

 duced TPN as measured by the reduction of oxidized glutathione via 

 a TPN-dependent glutathione reductase. The upper part of Table I 

 shows such an experiment. 



More recently attempts have been made to fractionate the protein 

 portion of this reaction mixture. The lower portion of Table I indi- 



TABLE I. Reduction of Oxidized Glutathione 



Reduced glutathione formed (micromoles) 



Complete reaction mixture* 1 . 3 



No TPN 0.4 



No GSSG reductase 0.4 

 No acetone powder extract, or heated 



extract, or egg albumen substituted 

 Ammonium sulfate fraction of ace- 

 tone powder extract 



0-0.2 0.6 



0.2-0.5 1.8 



0.5-1.0 0.7 



" The complete reaction mixture contains: acetone powder extract, 3.0 mg. 

 protein; ethanolic extract, 0.1 mg. chlorophyll; oxidized glutathione, 10 /zM; 

 TPN, 0.1 mM; MgClo, 1.0 mM; glutathione reductase. Time, 20 minutes; tem- 

 perature, 15°C.; illumination, incandescent light. 



* Supported by a grant from the National Science Foundation. Thanks are due 

 to Dr. E. Racker for a generous gift of purified glutathione reductase. 



285 



