267 



Sakae Katoh and Atuso Takamiya 



EFFECTS OF PLASTOCYANIN ON THE PHOTOREDUCTION OF 

 INDIGO CARMINE, FMN AND NADP IN THE PRESENCE OF 

 ASCORBATE AND DPIP 



Further investigations were carried out to test the effect of plastocyanin 

 in various oxidation reduction reactions of chloroplasts. First, it was found 

 that the rates of the Hill reaction with several electron acceptors were signifi- 

 cantly accelerated by the presence of a minute amount of plastocyanin. With 

 the Hill oxidants of rather high redox potential, such as cytochrome c and DPIP, 

 the observed acceleration may be explained by the simple assumption that 

 plastocyanin functions as an intermediary electron carrier in the reaction. It 

 could be rapidly reduced by the illuminated chloroplasts and then transfer its 

 electron to the Hill oxidants with high efficiency(8). 



In addition, the effect of plastocyanin on the photoreduction of reagents 

 with redox potentials lower than zero, such as indigo carmine, FMN and NADP, 

 was also studied. In this series of experiments, chloroplasts prepared from 

 fresh leaves of Brassica campestris (Komatsuna) were used. They showed a 

 rather low rate of Hill reaction with indigo carmine as the electron acceptor. 

 A significant high rate of photoreduction of indigo carmine, however, was ob- 

 tained upon the addition of a substrate amount of ascorbate and catalytic amount 

 of 2, 6-dichlorophenol indophenol. This is in accordance with the previously 

 published results of Vernon and Hobbs with chloroplasts from other plant 

 species^ ^^'. Now, the further addition of plastocyanin to such reaction mixture 

 resulted in a striking rise in rate of reduction of indigo carmine as shown in 

 Fig. 2. The degree of acceleration was a function of the amount of plastocyanin 

 and saturation was reached at about 0. 5 mjimole of plastocyanin added, thereby 

 indicating that the protein functioned catalytically. Plastocyanin itself was 

 entirely inactive in catalyzing the photoreduction of the dye in the absence of 

 chloroplasts. 



It will be seen from Table II that the activity for reduction of indigo 

 carmine per unit of chlorophyll decreased both on washing and hypotonic treat- 

 ment of the chloroplast. It is most probably due to a loss of a soluble factor 

 from the particulate system. The effectiveness of plastocyanin in stimulating 

 the reaction rate, however, was much more enhanced after these treatments. 

 Almost the same rate of photoreduction, on a chlorophyll basis, was obtained 

 with the three fractions in the presence of plastocyanin. It was inferred, there- 

 fore, that the chloroplasts requires plastocyanin as an essential component of 

 the photoreduction of indigo carmine in the presence of ascorbate and DPIP. 

 The data in Table II also show that plastocyanin is somewhat effective on the 

 slow photoreduction of indigo carmine in the absence of the ascorbate-DPIP 

 couple. Previously, Vernon and Hobbs noticed that the photoreduction of 

 indigo carnnine by chloroplasts was markedly stimulated on addition of the 

 supernatant fluid(18) This result may be ascribed to the possible presence of 

 plastocyanin in the soluble faction. 



