ATP Formation by Spinach Chloroplasts*t 



Andre T. Jagendorf and Joseph S. Kahn:|: 



Biology Depaytment and McCoUnm- Pratt Institute, 



The Johns Hopkins University, 



Baltimore, Md., U.S.A. 



The mechanism for conserving oxidation-reduction energy as ATP is 

 as much of a challenge in chloroplasts as it is in mitochondria. It is also a 

 matter of comparative biochemical interest to see how closely these two 

 mechanisms resemble one another. 



Our efforts in this area started when it became possible to study the 

 coupling between electron tiow and phosphorylation, thanks to the 

 discovery by Arnon and colleagues [i] that ATP is formed during ferri- 

 cyanide reduction in a Hill reaction. The rate of the Hill reaction is 

 stimulated by simultaneous phosphorylation, up to 3 • :; times under the 

 conditions of our experiments [2]. We observed that arsenate was an 

 uncoupler — increasing the rate of electron flow, while inhibiting phos- 

 phorylation — but only in the presence of ADP [3]. We have speculated 

 elsewhere [3, 4, 5] that this means either that a stable high-energy arsenate 

 intermediate is formed (analogous to the theoretical high-energv phosphate 

 intermediate in ATP formation); or that ADP is bound in a high-energy 

 complex first, and phosphate addition is the last step in ATP formation. 

 Since a stable high-energy arsenate intermediate seemed unlikely, we 

 suggested the alternative of a high-energy adenylate as the first step in 

 ATP formation. 



The evidence for this sequence, the reverse of that postulated for 

 mitochondria, was indirect. We have since been able to devise a more 

 direct experiment which indicates instead that a high-energv phosphate 

 intermediate is formed first, and ADP addition is the last step. Our later 

 conclusions are in accord with the conclusions to be drawn from recent 

 oxygen- 1 8 studies by Avron and Sharon [6] and by Schultz and Boyer [7]. 



The procedure for the more direct experiment [S] consists of illuminat- 

 ing chloroplasts in the presence of radioactive phosphate but without 

 ADP. A presumed high-energy phosphate intermediate (X ^ ^-P) has a 



* Contribution No. 329 from the McCollum-Pratt Institute, 

 t Supported in part by Research Grant RG3923 from the National Institutes 

 of Health. 



X Present address : Dept. of Botany, North Carolina State College, Raleigh, N.C. 



