266 



Sakae Katoh and Atusi Takamiya 



All the above-described features of the photooxidation of reduced plasto- 

 cyanin are in general agreement with those observed for the photooxidation of 

 reduced cytochrome c. A notable difference between the two photooxidizing 

 systems, however, was discovered when digitonin-treated chloroplasts were 

 fractionated with ethanol into a chlorophyll containing fraction and a soluble 

 fraction. Using a similar fractionation procedure, Nieman £t al. demonstrated 

 the necessity of a soluble factor, designated Factor 2 by them, for the photo- 

 oxidation of reduced cytochrome c by the chlorophyll bearing fraction' 1''. We 

 have confirmed this observation (Table I). However, the capacity for photo- 

 oxidation of reduced plastocyanin was not appreciably affected by the ethanol- 

 treatment and the addition of the soluble factor was without any effect on the 

 activity of the ethanol-precipitated fraction (Table I). Evidently, the photo- 

 oxidation system of reduced plastocyanin requires no additional soluble factor. 



TABLE I 



Effect of Soluble Factor on the Photooxidation of Reduced Plastocyanin 

 and Cytochrome C by the Ethanol-Precipitated Fraction 



/Ltmole oxidized 



Electron donors Addition mg chl. hour 



Reduced plastocyanin Mone 48 



Reduced plastocyanin Soluble factor 0. 4 ml 50 



Reduced cytochrome c None 3 



Reduced cytochrome c Soluble factor 0. 2 ml 15. 6 



Reduced cytochrome c Soluble factor 0. 4 ml 25. 6 



On the other hand, it was discovered that plastocyanin can replace the 

 soluble factor in accelerating the photooxidation of reduced cytochrome c by 

 the ethanol precipitated fraction. The reaction rate is increased by the addition 

 of plastocyanin to the level of photooxidation of reduced plastocyanin with the 

 same preparation. This stimulating effect of plastocyanin occurs by a mech- 

 anism different from that with ammonium sulfate, since the acceleration ob- 

 served in the presence of both substances was far higher than the sum of the 

 rates observed when they were added separately (45. 6 with plastocyanin alone, 

 39. 1 with 1. 3 M ammonium sulfate, and 142 ^moles of reduced cytochrome c 

 oxidized per mg chlorophyll per hour with plastocyanin and ammonium sulfate). 



These findings strongly suggest that Factor 2 of Nieman et al. may be 

 plastocyanin. Presumably in the photooxidation of reduced cytochrome c by 

 the digitonin-treated chloroplasts, plastocyanin acts as an intermediary electron 

 carrier which is first oxidized by the photooxidizing system of chloroplasts and 

 then reduced by the reduced cytochrome c. 



