128 Discussion 



phosphate compounds. The same appHes to calcium. If calcium is 

 added, it will compete with magnesium and will form an ATP complex, 

 which is not available, and there will be a loss of respiratory control 

 due to the addition of calcium. Also, one can then get the interesting 

 effect which Prof. Chance obtained, i.e. by adding small amounts of 

 calcium, respiratory control is only temporarily lost, and by adding more 

 calcium one gets complete loss of respiratory control. This could be 

 accounted for if some of the ATP is made unavailable or more slowly 

 available, due to a shift of equilibrium by the addition of calcium ; and 

 if more calcium is added, the equilibrium of available ATP is upset so 

 that there is a complete loss of respiratory control. 



Holzer : Prof. Chance explained his experiment with yeast and glucose 

 by the assumption that the ATP concentration in starved yeast cells is 

 very low, and that the ADP concentration is high. We have measured 

 ADP and ATP concentrations in yeast cells (Holzer, H., Witt, J., and 

 Freytag-Hilf, R. (1958). Biochem. Z., 329, 467). Most surprisingly, we 

 found that the concentration of ATP in aerobic starved yeast is as high 

 as in glucose-oxidizing yeast cells. If glucose is added under aerobic 

 conditions to starved yeast, the following is observed (Holzer, H., and 

 Freytag-Hilf, R. (1959). In preparation): during the first 7 seconds 

 ATP decreases very rapidly, increases again, and then a steady state 

 concentration is achieved, which is approximately as high as the steady 

 state concentration prior to glucose addition. The time during which 

 these changes in concentration occur is about 10-20 seconds, depend- 

 ing on the temperature (we measured at 25°). The ADP changes are the 

 exact reversal of those of ATP. From this it results that in starved 

 yeast the total concentration of ATP (without respect to compart- 

 mentation) is as high as in the steady state of glucose-oxidizing yeast. 



Chance : We do agree in certain respects, but it is important to point 

 out that Prof. Holzer and I measure nucleotide concentrations at dif- 

 ferent places. Also, we pretreat the yeast with a low ethanol concentra- 

 tion to obtain a minimal ADP concentration and then add glucose. The 

 ADP that we measure is that ADP accessible to the mitochondria, and 

 its rise in concentration upon glucose addition stimulates respiration. 

 The nucleotide that Prof. Holzer measures is the sum of the cytoplasmic 

 and mitochondrial concentration i.e. not only is there generation and 

 utilization of ATP on addition of glucose but there is also a movement 

 of ATP from the cytoplasm to the mitochondria. 



Slater : Prof. Chance, did you measure the K^ for ADP in the presence 

 of inorganic phosphate, and was the Kf„ for inorganic phosphate 

 measured in the presence of ADP? 



Chance : Yes ; we gave the K^^^ for ADP in presence of substrate and 

 phosphate, so that it was the dynamic Kfn referring to maximal respira- 

 tory activity. The K^ for phosphate was determined in the presence of 

 ADP and substrate. 



Lipmann: Did you wait long enough to obtain a steady state? The 

 3 2p equilibration in mitochondria takes a long time. 



Chance: Ours were steady state values, i.e. we waited long enough 

 until either maximal respiration or maximal change of the respiratory 



