PIIOSPIIOKYLATIOX BY ISOLATED CIILOUOPLASTS 343 



TABLE I. Flavin Mononucleotide (FMN), Ascorbate, Vitamin K, and Mag- 

 nesium Tun ;is (^ofactors of Photosjnthctic Phosphorylation by Broken Chloro- 



plasta 



f.'onditiona 



(A) No cof actors added 



(B) 10 mM Mg + + + 10 M^I ascorbate 



(C) (B) + 0.01 mM FMN 



(D) (B) + 0.03 M^NI vitamin Kj 



(E) (B) + 0.01 mM FMN + 0.03 mM vitamin K^ 



(F) (B) + 0.03 mM FMN 



(G) (B) + 0.3 nM vitamin Kj 

 (H) (B) + 0.03 mM FMN + 0.3 AtM vitamin K5 



(I) 10 fxM Mg + ^, 10 mM ascorbate, 0.1 mM FMN, 20.0 



. 03 ixM vitamin K5 



(J) (I), omitting Mg + + 5.9 



(K) (I), omitting ascorbate 2.9 



" Only at the low concentrations (arbitrarily chosen) of FMN and vitamin K.=, 

 could an interaction be demonstrated. At the high concentrations no interaction 

 was observed, indicating that the phosphorylation was then limited by some 

 factor other than FMN or vitamin Ki. 



The reaction mixture included, in addition to the above, 40 juM tris (hvdroxv- 

 methyl) aminomethane, pH 7.4, 20 mM K phosphate, pH 7.4, 20 fiM A^IP, pH 

 7.4, and broken chloroplasts containing 0.5 mg. chlorophyll. The volume was 

 made to 3 ml. with water. The reaction was carried out under nitrogen at 15°C. 

 in an illuminated Warburg respirometer with continuous shaking. Phosphoryl- 

 ation was determined as previously described (2,5). 



The amounts of FMN and vitamin K5 needed indicate that they func- 

 tion as catalysts and not as substrates. Ascorbate, although supplied 

 in larger amounts, is also used as a catalyst (3). The addition of pyri- 

 dine nucleotides did not affect the phosphorylation by the broken 

 chloroplasts, although it was found that most of the pyridine nucleo- 

 tide contained in Avhole chloroplasts is leached out on treatment with 

 water (7). 



Broken chloroplasts, like whole chloroplasts, were unable to carry 

 out oxidative phosphorylation in the dark with Krebs cycle inter- 

 mediates or reduced pyridine nucleotides. This again indicates that 

 the ATP synthesis by chloroplasts represents an anaerobic synthesis 

 of pyrophosphate bonds at the e.xpense of light energy by an enzyme 

 system peculiar to photosynthesis. 



