204 GIOVANNI FELICE AZZONE 



Azzone: We have not yet conclusive evidence about the chemical reaction 

 requiring the investment of energy. However, even if it is assumed that the ATP 

 stimulation is the consequence of a structural effect we still have to account for 

 the formation of a high energy intermediate, controlling the oxidation of suc- 

 cinate which precedes the mechano-chemical utilization of ATP. 



HoLTON : We must consider the availability of the succinate to the succinic 

 hydrogenase besides the possibility that its oxidation requires an investment of 

 energy. 



AzzoNE : According to the mechanism we have proposed, in non-phosphorylat- 

 ing preparations it is possible for the electron derived from succinate to proceed 

 through a sequence of electron carriers each of which possesses a higher redox 

 potential than the preceding one ; on the contrary, in intact mitochondria there is a 

 thermodynamically unfavourable step which must be circumvented by the invest- 

 ment of energy. 



Williams : I should like to say that, as Dr. Azzone is by now aware, we have 

 found, (a) that cysteine sulphinic acid reactivates rat liver mitochondria under 

 conditions as similar as we could get to the ones described in his paper in Nature, 

 (b) that, although pyruvate affords some degree of protection, a-ketoglutarate does 

 not, although it does maintain its substrate level phosphorylation. However, I think 

 it may be better to emphasize the measure of our agreement and to suggest that 

 you are now coming very close to saying that oxaloacetate is necessary. I think our 

 disagreement is then not so great and we can leave open the details of how oxalo- 

 acetate exerts this inhibition and how ATP relieves this inhibition. In Tyler's 

 work ATP relieved the inhibition without altering the oxaloacetate concentration, 

 so looking for PEP may not help us, and here I had wondered, as had Dr. Holton, 

 whether the ATP acts by segregating the oxaloacetate from the succinic dehydro- 

 genase, although there are no major optical density changes during this process. 



Slater : I should like to support what Dr. W^illiams said a moment ago, that a 

 very important point to look at is how ATP reverses oxaloacetate inhibition and I 

 should like to bring over a suggestion of my colleague Dr. Hiilsmann, that you 

 look into the possibility that ATP is activating oxaloacetate removal by reacting 

 with endogenous substrate and forming acetyl-coenzyme A which promotes the 

 removal of oxaloacetate; this is based on some recent experiments and explains 

 quite a lot of phenomena including the Amytal experiments, in the presence of 

 which oxaloacetate is not formed. 



AzzoNE : We have not been able to demonstrate the presence of oxaloacetate 

 in the arsenate-dicoumarol preincubated mitochondria so one must postulate that 

 oxaloacetate is compartmentalized in such a way that it cannot be reached by 

 chemical analysis. 



Mitchell : I should like to raise a point that may bring together Dr. Slater's 

 views and those of Dr. Holton. The suggestion that I am going to make comes 

 from work on micro-organisms which Dr. Moyle and I have been doing recently. 

 It so happens that in micrococci you can show that the oxidation of succinate can 

 be blocked by DNP when the membrane is intact. It can also be blocked by 

 mercury compounds or by arsenite. But, it can be demonstrated that the blocking 

 of oxidation by these inhibitors is not a direct effect on the oxidation system but is 

 due to blockage of the system by which "succinate" passes into the cell. If you 



