ATP FORMATION BY SPINACH CHLOROPLASTS 457 



TABLE I 



Formation ok [''-PJ-ATP after Illumination with ''-P 



All flasks received 8 min. pre-illumination with cold phosphate. Numbers refer 

 to protocol shown in Fig. i. The illumination indicated is the second one, of 15 sec. 

 duration, in the presence of ''-P. Reaction mixture contained 0013 M tris pH 80, 

 0-0033 ^^ ^IgClo, 0-033 M NaCl, 0-00003 ^i phenazine methosulphate, 0-00033 ^^ 

 phosphate, and chloroplasts containing from i to 5 /mioles of chlorophyll; total 

 volume 12 ml. 



A trivial possibility in this sort of experiment would be the formation, 

 in the light, of first a very small amount of labelled ATP, and then a larger 

 amount of a compound on a side pathway (such as carbamyl phosphate, 

 or other high-energy phosphate compound). This secondary product 

 might be the storage site for high-energy phosphate, and pass it on to the 

 large amounts of ADP added after illumination. This sequence would be 

 represented by equations 13 : 



X - -^^P + ADP -^ [=^-P]-ATP + X (i) 



PPJ-ATP + Y ^ ADP + Y - 3->p (2) 



Y - 3-P + ADP ^ Y + [3-^P]-ATP (3) 



If this mechanism were operating the specific acti\ity of phosphate in 

 the secondary product (Y ~ ^-P), and therefore the total amount of 

 p-P]-ATP formed in Reaction 3 v>ould be sensitive to variations in the 

 amount of unlabelled ATP, or in specific activity of ATP present before 

 adding ADP in the dark. This is not the case, however. 



The internal content of free ATP in chloroplasts is of the order of 

 200 400 m/Ltmoles/mg. chlorophyll. It can be removed almost completely 

 by breaking the plastids open in water. Whether the internal (unlabelled) 

 ATP is present or absent, i m/unole of [-^-PJ-ATP is formed mg. chloro- 

 phyll, owing to pre-illumination followed by ADP in the dark. 



A second indication of the absence of a simple dark equilibrium 

 between our stored intermediate and ATP lies in the constant amount of 

 p-P]ATP formed due to light, over a ten-fold variation in specific activity 

 of internal ATP due to dark reactions. Whether the dark controls have 



