264 



Sakae Katoh and Atusi Takamiya 



130 - 160 chlorophyll molecules per atom of copper, the copper in plastocyanin 

 can account for about half of the total coppex in the chloroplasts. It is note- 

 worthy that the concentration of plastocyanin expressed on a chlorophyll basis 

 is similar in magnitude to that of cytochrome f_'^^'. 



It was found that hypotonic treatment of whole chloroplasts resulted in the 

 release of a significant portion of plastocyanin. The grana fraction also con- 

 tained plastocyanin at a concentration comparable to that in the whole chloro- 

 plast. The grana also lost some plastocyanin by hypotonic treatment, thus 

 indicating the association of the protein with the subparticulate fraction of the 

 chloroplasts. All these findings strongly suggest that the protein under inves- 

 tigation is an integral part of the photosynthetic architecture of chloroplasts. 



PHOTOREDUCTION OF PLASTOCYANIN BY CHLOROPLASTS 



When oxidized plastocyanin was incubated with chloroplasts in the light, 

 a steady decrease in absorbance at 597 m^ was observed' ^^'. Upon addition 

 of ferricyanide at the end of the reaction, the blue color was restored to the 

 original level, thereby indicating that the decrease in absorbance was corre- 

 lated with reduction of the protein. Neither reduction nor oxidation of plasto- 

 cyanin was observed during incubation with chloroplasts in the dark. 



The rate of photoreduction usually obtained ranged from 100 to 200 

 /imoles protein per mg chlorophyll per hour(13), xhe rate increased with light 

 intensity and reached saturation at about 10, 000 lux. The reaction was optimal 

 between pH 7. 5 - 7.9. The reaction was completely inhibited by 10"^ M DCMU. 

 o-Phenanthroline and hydroxylamine were also potent inhibitors of the photo- 

 reduction. Cyanide, azide and arsenite were without effect at a concentration 

 of 10"^ M. The rate of photoreduction was increased by the presence of MgCl2 

 and ADP (the reaction system contains a sufficient amount of phosphate), indi- 

 cating that the reaction is coupled with phosphorylation. A very marked stimu- 

 lation of rate of reduction was observed on addition of ammonium sulfate, 

 which is known to uncouple the photophosphorylation from the electron trans- 

 ferring system of the chloroplasts^ ^'*). It may be concluded from these observa- 

 tions that plastocyanin is reduced by illuminated chloroplasts by the same 

 pathway as that of photoreduction of ferricyanide and KADP. 



Photooxidation of Reduced Plastocyanin by 

 Digitonin-Treated Chloroplasts 



It was found that reduced plastocyanin was readily photooxidized by 

 chloroplasts which had been treated with digitonin. A similar chloroplast 

 preparation was shown by Nieman and Vennesland to catalyze the photooxidation 

 of reduced cytochrome c^^^'. The time course of the photooxidation of reduced 

 plastocyanin by digitonin-treated chloroplasts measured by the increase in 

 absorbance at 59 7 m^ is illustrated in Fig. 1. No change in oxidation reduction 

 state of the added plastocyanin was observed in the dark. The removal of 



