632 LIGHT AND LIFE 



phyll. Faster rates of nucleotide reduction have been reported by 

 Hendley and Conn (13), who used chloroplast fragments and the 

 TPN-glutathione reductase from wheat germ or parsley. Recently, 

 Vishniac (25, 26) has reported a light-dependent reduction of TPN 

 in a cell-free system. In these experiments, as well as those of Hendley 

 and Conn (13) , the formation of TPNH was demonstrated indirectly 

 by measuring the formation of reduced glutathione in the presence of 

 glutathione reductase. In these two reports, the rate of TPN reduc- 

 tion was about 20 y^moles per hour per mg chlorophyll (i.e., about 

 40 /xmoles GSH formed per hour per mg chlorophyll) . 



At about this same time, Jagendorf (14) demonstrated that puri- 

 fied chloroplasts can reduce TPN but not DPN. Both TPN and 

 DPN were reduced when grana were used in place of chloroplasts. 

 In the presence of grana, the rate of reduction of DPN was about 6 

 times that observed with TPN. It should be noted that the experi- 

 mental procedure used in these experiments was similar to that em- 

 ployed by other investigators, namely, coupling the photochemical 

 reaction with a suitable dehydrogenase or reductase and measuring 

 the formation of the product of the coupling system. In the absence 

 of the coupling system, there was no measurable reduction of pyridine 

 nucleotides with either chloroplasts or grana. 



In 1956, San Pietro and Lang (21) presented evidence that under 

 the proper conditions photochemical reduction of pyridine nucleo- 

 tides can indeed be demonstrated directly. It was shown that when 

 DPN is incubated with chlorophyll grana in the light, either aerobi- 

 cally or anaerobically, DPNH accumulates in the medium. Similar 

 results were obtained with TPN. In these studies, however, it was 

 necessary to employ fairly high concentrations of grana and DPN. At 

 low grana concentrations (less than 60 micrograms chlorophyll per 

 ml) no measurable reduction of DPN occurred. At higher grana con- 

 centrations, the amount of DPNH formed was almost directly pro- 

 portional to the amount of grana initially present. 



Preliminary results at that time indicated that it was possible to 

 demonstrate reduction of pyridine nucleotides at low grana concen- 

 tration, provided that a soluble extract of chloroplasts was added. 

 This finding allows direct measurement of the initial rate of photo- 

 chemical reduction of pyridine nucleotides, since the reduction can 

 be followed spectrophotometrically by measuring the increase in ab- 

 sorption at 340 mju, of the reaction mixture without prior removal 

 of the chloroplasts. 



Soon thereafter, Arnon, Whatley, and Allen (3) reported evidence 



