ANTHONY SAN PIETRO 633 



in agreement with our findings. They demonstrated that the reduc- 

 tion of TPN by iUuminated chloroplasts was made possible by the 

 addition of an aqueous extract of whole chloroplasts. The reduction 

 process was not specific for TPN, since DPN was also reduced but at 

 a slower rate. 



Several years ago, San Pietro and Lang (22) reported the isolation 

 from either chloroplasts or whole spinach leaves of a soluble enzyme 

 which is required in addition to either chloroplasts or grana for the 

 photochemical reduction of pyridine nucleotides. The enzyme was 

 partially purified and certain of its properties studied. It was shown 

 that the enzymatic reaction requires the intact dinucleotide struc- 

 ture and, in addition, was more active toward TPN than toward 

 DPN. At that time, it was suggested that the enzyme be named 

 photosynthetic pyridine nucleotide reductase (PPNR) . 



More recently, the PPNR has been purified more extensively and 

 has been shown to be specific for TPN (19). Under the proper con- 

 ditions it has been possible to obtain reduction of TPN by illuminated 

 chloroplasts at a rate of about 180 /Amoles per hour per mg chlorophyll. 



The apparent lack of specificity of either the partially purified 

 PPNR or the aqueous extract of chloroplasts has been attributed, 

 both by Arnon et al. (3) and ourselves (22) , to the presence of 

 pyridine nucleotide transhydrogenase (5) in these preparations. Evi- 

 dence consistent with this explanation has recently been presented by 

 Keister and San Pietro (15) . It was demonstrated that the reduction 

 of DPN, in the presence of illuminated grana, requires both PPNR 

 and transhydrogenase; whereas, the reduction of TPN requires only 

 PPNR. It is possible to obtain similar results when chloroplasts are 

 used in place of grana (16). 



The transhydrogenase from spinach leaves has been purified ex- 

 tensively and certain of its properties studied (16) . In addition, a 

 possible role for this enzyme in photosynthesis has been proposed. 



The present paper is concerned with the purification and proper- 

 ties of the photosynthetic pyridine nucleotide reductase (PPNR) . 



Determination of Activity 



Enzymatic activity was determined by measuring the initial rate 

 of formation of reduced pyridine nucleotides in the Hill reaction. In 

 this procedure the photochemical reduction process is followed di- 

 rectly by measuring the increase in optical density at 340 m/i in the 

 following way. The reaction mixture, which contains buffer, pyridine 

 nucleotide, enzyme, and chloroplasts, is prepared in a glass cuvette 



