460 ANDRE T. JAGENDORF AND JOSEPH S. KAHN 



Discussion 



Lehninger : I guess I do not need to say how delighted I am to hear about the 

 occurrence of the ATP-ADP exchange in photosynthetic phosphorylation. This 

 finding of course establishes continuity between oxidative and photosynthetic 

 phosphorylation. The inhibition by ammonium ion is very interesting and I 

 wonder if you have any evidence for a requirement of or an inhibitory effect of 

 either potassium or sodium. 



Jagendorf : No, we haven't tested that. We are suspicious of the significance 

 of ammonium inhibition because it occurs with the highly purified enzyme without 

 any chloroplasts present. The dinitrophenol sensitivity of your ATP-exchanging 

 enzyme requires both the presence of the particles and re-coupling back into them, 

 which is much more suggestive. 



Lehninger: Well, of course, DNP and ammonium ion do not necessarily 

 uncouple at the same site. 



Baltscheff-Sky : I have two questions. First, was the light TCA control always 

 higher than the dark TCA control ? Second, did you use arsenate instead of 

 phosphate and did you in that case obtain an intermediate ; if so, was it more stable 

 or less stable than the phosphate intermediate ? 



Jagendorf: The light TCA control will always be a little bit higher than the 

 dark TCA control because in the light when '^"P is present, all one needs is a trace 

 of residual ADP, or a little break-down of the large amount of ATP present to 

 ADP, and ^-P-ATP will be formed. By being careful in eliminating ADP from the 

 system and especially by using short times to prevent ATP break-down, we can 

 keep this control down to a minimum. Also, if there were a slow exchange reaction 

 you would see it in the control where TCA is added after the light but before 

 ADP; I think that the fact that we have a 15-sec. exposure rather than several 

 minute exposure helps a lot in that respect. 



Arsenate does inhibit somewhat if you put it in with the phosphate but whether 

 it forms an intermediate or not I don't know. Even if arsenate did form an inter- 

 mediate we would not be able to trap it in a stable compound analogous to trapping 

 the high energy phosphate intermediate as ATP. 



Vennesland: Would you care to comment, Dr. Jagendorf, on the mechanism 

 of the arsenate efTect that you observed previously ? I am referring to the ATP- 

 dependent stimulation of ferricyanide reduction by arsenate. This appeared to be 

 a real and rather striking phenomenon, and I wonder how you would explain it ? 



Jagendorf : Well yes, it is a real phenomenon ; at that time we had only thought 

 of two possibilities : (a) that there was a stable high energy arsenate intermediate, 

 or {b) that ADP comes first. Now we find that ADP doesn't come first but another 

 possibility has occurred to us since ; perhaps the chloroplasts have membranes, not 

 the external membrane but ones surrounding the actual site of phosphorylation. 

 If penetration of arsenate or phosphate were to require the presence of ADP that 

 would explain the ADP requirement for arsenate uncoupling. However, when we 

 broke chloroplasts up into particles, uncoupling by arsenate still required ADP. 

 This experiment seems to argue against the concept of arsenate entry, which 

 leaves us now with the feeling that there may very well be a stable high-energy 

 arsenate intermediate bound to the enzymes. 



