122 Discussion 



DISCUSSION 



Backer: Prof. Chance, I noticed that all your experiments were 

 carried out at a very low level of total adenine nucleotide concentration, 

 i.e. 3 X 10-* M, which is far below the intracellular concentration. I 

 wonder whether the very sharp respiratory control which you have 

 observed could perhaps be an artifact due to the low concentration of 

 adenine nucleotide which you use. If one measures the ATPase activity 

 of mitochondria in the presence of an ATP-regenerating system with 

 phosphoenoZpyruvate and pyruvate kinase which remove inhibitory 

 ADP, one can observe very much higher levels of ATPase activity than 

 with the conventional method. We believe that an ATPase activity may 

 well be functional during oxidative phosphorylation provided that the 

 ATP is not channelled into some more useful purposes. We find that 

 when we measure respiration in liver mitochondria we observe little or 

 no oxygen uptake in the absence of ATP, but on addition of ATP even 

 without any acceptor we observe considerable uptake of oxygen. Of 

 course, there is further stimulation of respiration on the addition of a 

 phosphate acceptor such as glucose and hexokinase or on the addition 

 of ATPase. I wonder whether the respiratory control in the cell is 

 really as complete as you have proposed. Sir Hans has emphasized 

 many times that the Krebs cycle (or citric acid cycle, as he calls it) is not 

 only providing energy by oxidative phosphorylation but it participates 

 also in synthetic processes such as the formation of glutamic acid. We 

 could visualize here a release mechanism due to ATPase which allows 

 for a certain amount of substrate to be getting through the Krebs 

 cycle, even under conditions of overproduction of ATP. 



Chance : I presented the yeast experiment because it showed that, in 

 cells of this type, we did not get more than a 50 per cent decrease in 

 respiratory rate, at a time when we observed a marked drop of intra- 

 cellular ADP. If we use ascites tumour cells, they show a much more 

 dramatic drop (over tenfold, see p. 119). 



Backer: But even in the ascites cells, the complete standstill of 

 respiration after addition of glucose is very brief; at least in our experi- 

 ments it lasts only two or three minutes and then respiration is resumed. 

 In other words, the complete inhibition of respiration is only short- 

 lived. 



Chance: This blockage affords an excellent way of studying the 

 mechanism of control of oxidative metabolism. On a quantitative basis, 

 the Qoj at 26° in the blocked state is 2-6 and is stable for over 10 minutes. 

 In the rapid phase the Qoj is 20-30. Thus, a fairly active metabolism for 

 this type of cell proceeds in the "blocked" state. 



Hess : In order to illustrate the significance of this blocking of oxida- 

 tive metabolism I shall give some quantitative chemical data, first on 

 the critical stationary ADP concentration of resting mitochondria in the 

 blocked state 4 and, secondly, as applied to intact cells, on the glucose- 

 oxygen stoichiometry in ascites tumour cells. The ADP and ATP con- 

 centration in resting mitochondria (state 4) was measured after depro- 

 teinization, by means of enzymic tests, ADP by using the pyruvate 



