230 



C. C. Black and A. San Pietro 



240 280 



320 360 400 



WAVELENGTH m/. 



440 480 



350 450 



WAVELENGTH, m/i 



Fig. 1. (Left) Lower portion: absorption spectra of spinach 



phosphodoxin. Solid line, 0. 1 N NaOH; broken line, 0. 1 N HCI. 



Upper portion: difference spectrum. 



Fig. 2. (Right) Activation and fluorescence spectra of spinach 



phosphodoxin. 



BIOLOGICAL CHARACTERISTICS 



The endogenous spinach chloroplast fragment photophosphorylation (0. 5 

 to 3 umoles of ATP/mg chlorophyll/hr) is stimulated over 200-fold by the 

 addition of spinach phosphodoxin (Fig. 3). As indicated in Fig. 4, this 

 aerobic reaction is linear for short periods of illumination. 



In an earlier publication ^^^^ the reaction was only 60 per cent inhibited 

 under nitrogen. Further work with prepurified nitrogen indicates that the 

 reaction with spinach chloroplast fragments definitely requires aerobic 

 conditions (Fig. 4). It should be noted that this is in contrast to the photo- 

 phosphorylation catalyzed by Rhodospirillum rubrum phosphodoxin with R. 

 rubrum chroma tophore fragments, which is unaffected by anaerobiosis^ ' >. 



Photophosphorylation with spinach chloroplast fragments plus spinach 

 phosphodoxin has been shown: to be linear with chlorophyll up to 40 ^grams 

 of chlorophyll per ml; to have a pH optimum in Tris-HCl buffer between 7. 4 

 and 7.8; to require a divalent ion for maximum activity; to respond in a 

 sigmoid fashion to increasing intensity of white light with a distinct lag up to 

 100 foot-candles, reaching saturation between 1000 and 2000 foot-candles; 

 and to be unaffected by PPNR, pyridine nucleotide transhydrogenase, and the 

 antibody to the transhydrogenase. From Fig. 5 it can be seen that photo- 

 phosphorylation catalyzed by spinach phosphodoxin is sensitive to the usual 

 inhibitors of photophosphorylation. In addition to these inhibitors 50% 



