636 LIGHT AND LIFE 



The nucleotide specificity of the enzyme in the presence of iHumi- 

 nated chloroplasts is ilkistrated by the data presented in Table 2. It 

 can be seen that TPN and acetylpyridine-TPN are reduced in this 

 system, whereas neither DPN nor acetylpyridine-DPN are reduced. 

 In separate experiments, it has been established that neither nicotina- 

 mide mononucleotide (NMN) , nicotinamide riboside (NR) nor the 

 a-isomer of DPN is reduced in this system. These findings are con- 

 sistent with both the postulation of Arnon et al. (3) that TPN is 

 a catalyst of photosynthetic phosphorylation (cf. Arnon, this sym- 

 posium) and the observation of Jagendorf (14) that chloroplasts can 

 reduce TPN but not DPN. 



It should be noted that it is possible to demonstrate the reduction of 

 DPN in this system provided transhydrogenase is added and the 

 concentration of DPN is about 3-4 X 10"^ ^ (16). 



TABLE 2 

 Nucleotide Specificity 



Each reaction mixture contained 300 ^moles of Tris buffer, pH 7.5, nucleotide, 

 2 units of enzyme, and chloroplasts equivalent to 91 micrograms of chlorophyll. 

 Final volume was 3 ml. Illumination for 5 minutes at 24°C. 



Nucleotide Concentration ^Euo AE; 



365 



TPN 2X10-^M 0.78 



DPN 2X10-" A/ 0.01 



Acetylpyridine-TPN* 1 . 3 X 10"" A/ . 90 



Acetylpyridine-DPN* 1.3X10-='A/ 0.03 



* The millimolar extinction coefficient for the reduced acetylpyridine analogues 



is 9.1 (23). 



Independence oj Dye Reduction from Enzyme 



Our earlier results (22), as well as those reported here, indicate 

 that PPNR is required in addition to chloroplasts or grana for the 

 photochemical reduction of TPN. These data support the hypothesis 

 that the enzyme catalyzes the transfer of hydrogen (or electrons) 

 from the photolytic system to TPN. Additional evidence for this 

 hypothesis is provided by the finding that the enzyme is without effect 

 on the photolytic activity of chloroplasts or grana when measured 

 either spectrophotometrically with 2,3,6-trichlorophenol-indophenol 

 or manometrically with ferricyanide. The lack of dependence on 

 PPNR of dye reduction is illustrated by the data presented in Table 

 3. It is clear from these data that the rate of dye reduction observed 

 in these experiments is unaffected by the presence of PPNR at each 



