240 MARTIN KLINGENBERG 



Klingenberg: ADP cannot induce DPN reduction. The respiration is not 

 stimulated. 



Chance : We have been studying for some time the ADP-inhibition of succinate 

 oxidation. When you add ATP to mitochondria which are not too tightly coupled, 

 you may produce ADP and phosphate. Under these conditions I expect the same 

 as you have observed, cytochrome c goes oxidized, and respiration may be inhibited. 

 I think it is something which should be controlled in these preparations because 

 they are sensitive to small amounts of ADP which will inhibit respiration strange 

 as it may seem. 



While I certainly agree that ATP can reverse electron transfer, I do not know 

 whether you can do it in the presence of oxygen and thermodynamics speaks 

 against it. I do not question the phenomenon but I am not sure whether you have 

 demonstrated it to me at the cytochrome level. 



Two comments occur to me : first, when the mitochondria became anaerobic, 

 you report flavin slowly became oxidized. This may be hard to measure accurately. 

 The second point is that we get much more DPN reduction on adding ATP in the 

 presence of succinate, in fact there isn't just any more DPN to be reduced when 

 mitochondria go anaerobic with added ATP, but you only get 40°,,. Is this a differ- 

 ence in preparations ? 



Klingenberg : This may be ; we sometimes get 60% but never more ; I do not 

 think this is very significant. In the presence of ADP or phosphate, ATP does not 

 inhibit respiration and does not oxidize cytochrome c. This would be an argument 

 against the hypothesis that ADP is generated by the addition of ATP and thus has 

 the effect on cytochrome c. Also in the presence of DNP no oxidation of cytochrome 

 c or inhibition of respiration occurs on addition of ATP. I do not think that we 

 have observed an electron transport reversal in the cytochrome region. I think we 

 have observed a reversed interaction of ATP with the cytochromes which results 

 in an increase of the respiratory control. 



Chance : In the experiments I reported this morning, special precautions were 

 taken to exclude the possibility that the electron acceptor for the observed oxidation 

 of cytochrome c in the presence of ATP was oxygen. If it were oxygen, then 

 obviously no "reversed interaction of ATP with the cytochromes" could have 

 been proved. In Dr. Klingenberg's excellent experiments, which have just been 

 reported, oxygen was present and no inhibitor of the oxidase was added ; the system 

 being in a steady state. Thus, an ATP induced inhibition of electron transfer 

 would be sufficient to explain the observed results; there being no demonstration 

 that the acceptor of electrons from cytochrome c was a substance at a lower and 

 not at a higher redox potential the former would be required in reverse electron 

 transfer. 



Klingenberg : I had stated in my report that we interpret the oxidation of the 

 cytochromes c and a to demonstrate an interaction of the ATP with the cyto- 

 chromes by a reversal of oxidative phosphorylation. Although oxygen had not been 

 excluded, these experiments should show the reverse interaction of ATP by way 

 of oxidative phosphorylation reactions as clear as, in the opposite manner, an 

 influence of ADP and phosphate on the redox state of the cytochromes shows an 

 interaction by way of oxidative phosphorylation. This is further supported by the 

 increase of the respiratory control after addition of ATP. 



