DANIEL I. ARNON 499 



was preferentially reduced (17). A water-soluble factor (F) , readily 

 extractable from chloroplasts, was required for TPN reduction and 

 ^\•as found to be a jjrotein in nature (17) . In the jncsence of the 

 soluble protein, catalytic amounts of clilorophyll (0.1 micromolc) 

 reduced sidjstrate amounts of TPN (4 micromoles) . The accumulation 

 of TPN H.J was found to be (17) accompanied by an evolution of 

 oxygen (Table 1), in accordance with reaction /: 



light 

 2 TPN + 4 HoO 



chloroplasts -(- F 

 2 TPNHo + Oo 4- 2H2O 



(1) 



The TPN-reducing factor (F) has subsequently been purified by San 

 Pietro and Lang, who called it the photosynthetic pyridine nucleotide 

 reductase (133). They found the purified enzyme to be more active 

 in reducing TPN than DPN, as was observed with the chloroplast 

 extract by the Berkeley group (17) . 



It is clear, therefore, that cliloroplasts have an enzymatic appara- 

 tus necessary for the photochemical reduction of TPN that is coupled 

 with oxygen evolution. What remains to be determined is the source 

 of ATP in photosynthesis, or more specifically, the cellular site and 

 the mechanism by which ATP is formed during photosynthesis. 

 From the standpoint of cellular physiology, the important question 

 is whether the ATP used in photosynthesis is formed by some light- 

 driven assimilation of inorganic phosphate, catalyzed by enzymes 



TABLE 1 



Equivalence of TPN Reduction and Oxygen Evolution by Illuminated 



Chloroplast Particles in the Presence of 



Excess Chloroplast Extract (CE) 



(Arnon, Whatley, and Allen, 18) 



The reaction mixture included, (in addition to TPN) in a final volume of 3 ml, 

 "broken" chloroplasts (PiJ containing 0.1 mg chlorophyll; chlorophyll-free chloroplast 

 extract made from chloroplasts containing 2 mg of chlorophyll; tris-buffer, pH 8.3, 

 80 jumoles; and NaCl, 35 nm. The reaction was run to completion at 20°C under 

 nitrogen, at an illumination of about 35,000 Lux, with KOH in the center well of the 

 manometer vessel. After centrifuging off the particles, reduced TPN in the supernatant 

 fluid was measured spectrophotometrically at 340 m^. Oj evolution was measured- 

 manometrically. 



