Discussion 129 



carrier had been established. Prof. Lipmann's point was very well 

 taken with regard to phosphate : it does take a while to achieve a steady 

 state with phosphate. Perhaps it is almost two minutes before we get 

 the steady state. 



Slater : The K^ values which I quoted in answer to Dr. Hess' question 

 (p. 85) are the same -K^'s measured under exactly the same conditions. 

 The figures are very similar ; I mentioned a K^n of 70 (xm, corresponding 

 to your 56 (xm. 



Siekevitz: ADP must be added to mitochondria in order to get a 

 stimulation of respiration. Yet mitochondria always have ADP there 

 when we extract them with acid and measure the amount of ADP. 

 ADP, ATP and AMP are present, according to our information. We 

 think that the ADP is bound in the mitochondria and unavailable for 

 stimulating oxidation. We have never found, during oxidative phos- 

 phorylation, any increase or decrease in ADP or ATP within the mito- 

 chondria. They always seem to be at the same levels. It has been found 

 that they will take up inorganic phosphate ; i.e. the phosphate concentra- 

 tion within the mitochondria can be increased several fold (Bartley, W., 

 and Davies, R. E. (1954). Biochem. J., 57, 37; Siekevitz, P., and Potter, 

 V. R. (1955). J. bioL Chem., 215, 237). This finding might have some- 

 thing to do with the difference in the K^ values that you get, and also 

 with the lag period obtained during the equilibration when you add 

 phosphate, as against the addition of ADP when no lag period was 

 observed. ADP reacts immediately, because even if there is ADP in the 

 mitochondria, it is bound and unavailable; it might be there at just 

 below threshold level. If you add phosphate, you may have to increase 

 the phosphate concentration in the mitochondria to a certain level before 

 respiration is stimulated. 



Chance : You have put your finger on the central question of chemical 

 analysis of mitochondria and intact cells. Most analyses simply do not 

 show the concentrations of ATP /ADP which our K^ values suggest, 

 except for the data that Dr. Hess has presented (see Table I, p. 123). 



Hess : The criterion for this type of test is the state of mitochondrial 

 respiration. The mitochondria should scarcely respire in the presence of 

 substrate and endogenous ADP or added ADP after reaching the steady 

 state equilibrium of the resting condition which we call state 4 (ADP 

 being the rate-limiting factor). The respiration rate must be very small 

 in this state; in other words, the mitochondria must have respiratory 

 control. The data I have discussed (see Tables I and II, above) are ob- 

 tained through analysis of mitochondria in this state, which was de- 

 tected by means of the oxygen electrode. We have done this analysis 

 many times, and can also titrate the ATP down by adding tri-iodo- 

 thyronine (see Table II, above). I wonder whether Prof. Siekevitz has 

 kept his mitochondria in this state of respiratory control. Then the 

 discrepancy between his and our data would be understandable. 



CELL MEIAB. 



