594 LIGHT AND LIFE 



of Jagendorl (II, 4, 5), his collaborators, and others (15, 19, 9, 13), 

 have since elucidated many features of the process, although the exact 

 mode of ATP formation and TPN reduction is still an open question. 



In 1957 Marre and Forti (13), on the basis of inhibitor studies, 

 attempted to rule out the reductive dephosphorylation hypotheses 

 set forth by Kandler and by Strehler. Subsequently Avron and Jagen- 

 dorf (5) presented data which was interpreted as a lack of ATP-phos- 

 phate-exchange in illuminated chloroplasts in the presence of Hill 

 reagents, a result having pertinence to the above question. However, 

 even though it had been demonstrated that the Hill oxidant he em- 

 ployed (ferricyanide) was reduced through a coupled phosphorylation, 

 much as was TPN, it was not shown that pyridine nucleotide reduction 

 is unaccompanied by phosphate-ATP exchange. In the latter case the 

 potential might well have been expected to be determinative. 



The present studies were therefore undertaken in order to test more 

 critically the hypothesis set forth in 1951. It has been found that 

 there is indeed a light-induced incorporation of Pj^^ into added ATP 

 in the presence of TPN (and indeed of other Hill oxidants) and 

 spinach chloroplasts, amounting to as much as 30 per cent of the rate 

 of ADP photophosphorylation. The results indicate that the exchange 

 is largely the result of a dark ATPase activity followed by photo- 

 phosphorylation. 



Materials and Methods 



Spinach chloroplasts were prepared according to the method of 

 Jagendorf (11) in buffered sucrose solution (150 ml) by grinding 

 spinach leaves (30.0 g) , macerated with scissors after a prior 30- 

 minute illumination, for 1 minute in a chilled mortar and pestle. 

 After filtering through cheesecloth, debris was removed at 0°C by 

 centrifugation at 500 g for 1 minute, followed by 4 minutes at 2000 g 

 to remove the chloroplasts from fragments in the supernatant liquid. 

 They were then resuspcnded in sucrose medium (4 ml) , washed once 

 by centrifugation, suspended in 3 ml of sucrose medium, and kept at 

 0°C and in the dark until used. 



At zero time an appropriate aliquot was pipetted into a reaction 

 chamber illuminated by 2 20-watt fluorescent bulbs (2 inches be- 

 tween light and sample) at 15°C. The experiments were terminated 

 by adding an equal volume of b^/o perchloric acid to the mixtine. 

 Samples were chilled to 0°C. 



After 30 miiuites or longer, the solids were removed by centrifuga- 

 tion, the luuleotides were adsorbed onto activated charcoal, which 



