Photosynthetic Phosphorylation by Isolated 

 Spinach Chloroplasts* 



F. R. WHATLEY, M. B. ALLEN, and DANIEL L ARNON, Laboratory 

 of Plant Physiology, Department of Soils and Plant Nutrition, University of 



California. Berkeley 



In the light, but not in the dark, isolated chloroplasts esterified 

 inorganic phosphate (Pi) into adenosine triphosphate (ATP). This 

 light-dependent process has been termed "photosynthetic phosphoryl- 

 ation" (1). ATP accumulated (1,2) when illuminated chloroplasts 

 were incubated under nitrogen with the proper cofactors and a phos- 

 phate acceptor, either adenylic acid (AMP) or adenosine diphosphate 

 (ADP). The cofactors so far identified for photosynthetic phosphoryl- 

 ation by isolated chloroplasts are Mg++, ascorbate, flavin mono- 

 nucleotide (FMN), and vitamin K (3,4). The procedures for isolating 

 chloroplasts are described in detail elsewhere (5) . 



The product of the reaction was identified as ATP (a) directly, by 

 adsorption on Norite, followed by hydrolysis of the labile phosphate 

 and (b) indirectly, using glucose plus hexokinase as an ATP-acceptor 

 system, either during the process of photosynthetic phosphorylation 

 or following the termination of the reaction and the chemical isolation 

 of the ATP; in both cases the product of the hexokinase reaction, 

 glucose-6-phosphate, was identified chromatographically (2). 



The esterification of phosphate proceeded unimpaired under an at- 

 mosphere of purified nitrogen (3), showing that the presence of free 

 oxygen is not a prerequisite for the occurrence of photosynthetic 

 phosphorylation. In fact it was observed with whole chloroplasts that 

 the presence of air actually depressed the phosphorylation when 

 FMN and vitamin K were added as cofactors. 



The process of photosynthetic phosphorylation has been repre- 

 sented (2,6) by the equations 1, 2, and 3. 



chloroplasts 



^.' + HoO ►2[H]4-[0] (1) 



* Aided by grants from the National Institute of Health and the Office of 

 Naval Research. 



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