270 



Sakae Katoh and Atusi Takamiya 



EFFECT OF PHOTOREDUCTION OF NADP BY THE 

 DIGITONIN-TREATED CHLOROPLASTS 



Plastocyanin was also found to be effective in stimulating the photoreduc- 

 tion of NADP (Hill reaction), when digitonin-treated chloroplast fragments, 

 which had lost most of the activity in question, were used. The addition of a 

 catalytic amount of plastocyanin resulted in partial recovery of the lost activity. 

 A complete reactivation was obtained at a concentration as low as about 10"° 

 mole per 3 ml of the reaction mixture (Table IV). The stimulating effect ob- 

 served on addition of an aqueous extract of chloroplast to the digitonin-treated 

 chloroplasts may be also due to the presence of plastocyanin in the extract. 



TABLE IV 



Effect of Plastocyanin on NADP Photoreduction 

 by Digitonin-treated Chloroplasts 



fxmole reduced 



Preparation Addition nag chl hour 



(mole) 



Untreated chloroplast - 118 



Digitonin-treated - 



Chloroplast 0.13x10-8 4.9 



0. 33 x 10-8 7.8 

 0.65 X 10-8 10.5 



1. 30 X 10-8 11. 1 

 1. 95 X 10-8 11, 



PPNR is necessary for the reduction of NADP, no photoreduction of NADP is 

 observed when PPNR is omitted even in the presence of plastocyanin. Addition 

 of the following substances could not replace the role of plastocyanin in stimu- 

 lating the reaction in question; FMN, PMS, vitamin K3, ascorbate, ferri- and 

 ferrocyanide, benzoquinone, and cytochrome c. The Rhus blue protein, another 

 non-autooxidizable form of copper protein (kindly provided by Dr. Omura^^^'), 

 was also without effect. The degree of stimulation due to plastocyanin depends 

 on the light intensity. The effect was not very significant at light intensities 

 lower than 7, 000 lux. At this intensity the reaction rate without addition of 

 plastocyanin reaches saturation. With added plastocyanin, however, the 

 reaction rate increases with further increase in light intensity up to 40, 000 

 lux. The plastocyanin-induced photoreduction of NADP is sensitive towards 

 the inhibitors of the Hill reaction, such as DCMU and o-phenanthroline. This 

 fact indicates that the observed change involves the Hill reaction activity 

 including the oxygen evolving system. 



