522 LIGHT AND LIFE 



phorylating site can be readily envisaged in the span between TPN 

 and cytochromes (9) . 



Further evidence, derived from fractionating chloroplasts, will be 

 given in the next section for the conclusion that cyclic photophos- 

 phorylation catalyzed by phenazine methosulfate probably proceeds 

 by way of a "bypass" and is less dependent on enzymatic chloroplast 

 constituents than photosynthetic phosphorylation catalyzed by either 

 vitamin K or FMN. 



11. Structural Association of Chorophyll with the 

 Photophosphorylating System 



In photosynthetic bacteria the photophosphorylating system is struc- 

 turally bound to chlorophyll in the smallest particles that function 

 as units in the absorption of light energy, the chromatophores. Their 

 analogues in green plants are the grana. It was of interest, therefore, 

 to determine whether in chloroplasts photophosphorylation is indeed 

 localized in the grana. 



Photosynthetic phosphorylation was first observed in intact chloro- 

 plasts (Fig. 11), but experiments with disrupted chloroplasts soon 

 demonstrated that structural integrity was not essential for this 

 process. When whole chloroplasts were broken, active photophos- 

 phorylation systems were reconstituted by a recombination of chloro- 

 plast fractions and added cof actors (13, 173) . This technique proved 

 effective in investigating the mechanism of photophosphorylation 

 but provided no rigorous evidence that the site of photophosphoryla- 

 tion is in the grana. 



Direct evidence for the localization of photosynthetic phosphory- 

 lation in grana, freed from other chloroplast fractions, was obtained 

 by Miiller, Steere, and Arnon (110), who prepared purified grana by 

 sonication of isolated whole chloroplasts followed by a density gradient 

 centrifugation technique. The purity of the grana obtained by these 

 methods was determined by examination of electron micrographs of 

 freeze-dried and air-dried grana preparations (Figs. 12 and 13). The 

 freeze-drying technique avoids artifacts resulting from chemical fixa- 

 tion and retains the natural shape of the particles (142) . Cyclic 

 photophosphorylation by purified grana is shown in Table 5. 



Table 5 shows that, at the high light intensity at which cyclic photo- 

 phosphorylation by purified grana was measured, the highest rates 

 were obtained in the system catalyzed by phenazine methosulfate. 

 Photophosphorylation in this system was not increased by the addi- 

 tion of an aqueous (hlor()j:)last extract. By contrast, photophosphory- 



