342 F. R. WHATLEY, M. B, ALLEN, D. I. ARNON 



symbol [O] is intended to express the experimental observation that 

 phosphorylation proceeds in the absence of molecular oxygen (3,6). 



Photosynthetic phosphorylation by whole chloroplasts was shown 

 to be unaffected also by the presence or absence of carbon dioxide. 

 If carbon dioxide was eliminated from the reaction mixture the phos- 

 phorylation proceeded unimpaired (2). It was also shown that whole 

 chloroplasts fail to carry out any oxidative phosphorylation when sup- 

 plied with Krebs cycle intermediates (5). 



In earlier experiments active phosphorylation was obtained only 

 with whole chloroplasts ( 1 ) . It was found later that unbroken chloro- 

 plasts were not essential, provided that the proper cof actors were 

 added to the reaction mixture (7). Washed whole chloroplasts were 

 first isolated in 0.35 M NaCl and were then suspended in distilled 

 water. By this treatment their gross structure was destroyed. Photo- 

 synthetic phosphorylation by the broken chloroplasts was found to 

 he similar to phosphorylation by whole chloroplasts. The enzymes in- 

 volved in photosynthetic phosphorylation are apparently unaffected 

 by the water treatment. 



The light dependence of the photosynthetic phosphorylation 

 catalyzed by the broken chloroplasts is illustrated by the progress 

 curves in Fig. 1. All of the inorganic phosphate added to the reaction 

 mixture (20 juM) was used up in the light in 30 minutes, whereas vir- 

 tually no esterification was observed in the dark. With the broken 

 chloroplasts, the rates of phosphorylation commonly observed were 

 twice those obtained with whole chloroplasts, suggesting that some 

 permeability factor limits the rate with the latter. 



The phosphorylation by the broken chloroplasts, like that by whole 

 chloroplasts, proceeded unimpaired under anaerobic conditions, as 

 shown in Fig. 1 . This confirms the previous conclusion that free oxygen 

 is not involved in photosynthetic phosphorylation. 



The dependence of the broken chloroplasts on cofactors resembles 

 that of whole chloroplasts. Table I shows the results of an experiment 

 in which the known cofactors were added separately or in combina- 

 tion. Details of the optimal concentrations of the various cofactors 

 will be presented elsewhere. All these cofactors are known to be com- 

 ponents of green leaves (8), and some, like vitamin K, are char- 

 acteristically concentrated in the chloroplasts (9). 



It is likely that Mg++ has a catalytic role in the transfer of phos- 

 phate groups (10). The other cofactors may serve as electron carriers. 



