50 ALBERT L. LEHNINGER 



Discussion 



Estabrook: Dr. Fugmann and I have studied the reactions of the endogen- 

 ous pyridine nucleotides of the digitonin particles and we find that the extent of 

 reduction of the pyridine nucleotide during the steady state is largely dependent 

 on the presence of versene. In other words one cannot get a cyclic response of the 

 pyridine nucleotide with ADP without versene being there. Have you tried any of 

 these metal chelators or do you have any indication that the action of the M-factor 

 has any relation to chelating properties ? My second question is : according to your 

 second mechanism, phosphate itself should serve as an uncoupler because it will 

 liberate high concentrations of free carrier. I wonder if you have any explanation 

 for this point. 



Lehninger : With regard to your first question I should have mentioned that 

 for the action of the M-factor we need Mg-+ so possibly some function of the metal 

 is required here, but the effect is not given by EDTA. With regard to your second 

 question, I don't think that the mechanism I had on the board is the last word on the 

 mechanism of phosphorylation but you have to start somewhere with a working 

 hypothesis. The value of any of these hypotheses is, I think, just a matter of how 

 many crucial experiments you can design based on them. In the case of the 

 second mechanism, phosphate could act as an uncoupling agent only if P~M is 

 split by either hydrolysis or reaction with E, to regenerate free M, which is 

 required for respiration as well as the carrier. Since the molar amount of E is 

 limited, free M could be regenerated only if free E could be regenerated. Arsenate 

 can uncouple because the intermediate arsenylated compounds are presumably 

 unstable. 



Chance: I was very interested in Dr. Lehninger's discussion of the mechano- 

 protein which I think is a very important concept, especially in view of the work of 

 Pullman and Racker. However, being part physical chemist, there is a critical 

 question which I can ask, that is whether the time-course of contraction of the 

 hypothetical mechano-protein in mitochondria is one that is compatible with its 

 function in oxidative phosphorylation. It is true that the ADP-induced light 

 scattering increase on contraction, observed by Packer and myself, is a rapid one 

 and is possibly compatible with the mechano-protein idea, but it seems to me that 

 it is the opposite sense to the ATP-induced contraction, which also seems to me 

 to be on a slower time scale. Are there two kinds of mechano-proteins, one studied 

 in the presence of ATP and on a fairly long time scale, and one observed on ADP? 



Lehninger : In our earlier publications it appeared that ATP-induced contrac- 

 tion was quite slow; this was because we did not understand the optimum condi- 

 tions. Now on more refined investigation I am not sure that there is a great differ- 

 ence in the speed of ATP- and ADP-induced contraction. I don't think there is any 

 great disparity there, but there is no doubt that the changes that we and Packer 

 observed lag behind the changes in the steady state of the carriers. However, I 

 don't think this is completely mcompatible with the picture I have drawn for the 

 following reason : the morphology of the mitochondrion is pretty complicated, and 

 it is possible that in ATP- or ADP-induced contraction there is a sequence of 

 events. First, in the primary event a given molecule changes shape or contracts in 

 the presence of ATP, possibly synchronous with change in oxidation-reduction 



