292 Discussion 



measured. We observe that low ammonia concentrations stimulate 

 respiration but higher concentrations inhibit. The effect upon the cyto- 

 chromes is a striking one. Whereas in the absence of ammonia, cyto- 

 chrome c (551-540 m|ji) is very little reduced in the steady state, low 

 concentrations of ammonia cause reduction to the point where cyto- 

 chrome a (605-650 mfi) is also affected, with the consequent reduction 

 of respiratory rate. It is our conclusion that ammonia causes an un- 

 known type of inhibition of electron transfer in the respiratory chain. 

 Furthermore, the increased reduction of pyridine nucleotide (340- 

 374 mjx) shows clearly that it is not lack of substrate that causes the 

 respiratory inhibition; more DPNH is present in the presence of 

 ammonia than in the absence of it. In view of these results, it seems 

 scarcely reasonable that the effect of NH4+ upon the a-ketoglutarate 

 equilibrium is the sole cause of respiratory inhibition. 



On the other hand, the effects of ammonium chloride upon the steady 

 state level of pyridine nucleotide in mitochondria which are freely 

 permeable to this ion, have been studied (Chance, B., and Williams, 

 G. R. (1956). Advanc. Enzymol, 17, 65) and it is found that a system 

 buffered with a-ketoglutarate-glutamate "redox buffer" will respond 

 to m-molar concentrations of ammonium chloride with an oxidation of 

 reduced pyridine nucleotide and an inhibition of respiration. It is 

 apparent, however, from the data presented in Fig. 2 that the metabolic 

 regulation in the intact yeast cell is a more complex one and is probably 

 inadequately explained by the hypothesis presented by Prof. Holzer. 



Holzer : The changes in the rate of oxygen uptake, as measured after 

 the addition of ammonium ions by Prof. Chance, are in accordance with 

 our experiments: we worked with approximately 80 m-moles NH4+ 

 per litre, i.e. in a concentration range in which Prof. Chance also ob- 

 served an inhibition of oxygen uptake. The results concerning the state 

 of reduction of DPN are in contrast to our findings : from analyses of the 

 concentrations of alcohol and acetaldehyde we have calculated a 

 diminution of the ratio DPNH /DPN after the addition of NH4+ ions 

 (Holzer, Holzer and Schultz, 1955, loc. ciL; Holzer, Schultz, and Lynen 

 1956, loc. ciL), whereas Prof. Chance observes an increase in the ratio 

 DPNH /DPN. This discrepancy might be due to the following: (1) our 

 experiments were carried out at pH 6, whereas Prof. Chance works 

 under alkaline conditions ; (2) we measured — via the determination of 

 alcohol and acetaldehyde by means of the alcohol dehydrogenase equilib- 

 rium — the ratio of the concentrations of the free dissociating pyridine 

 nucleotides, whereas, Prof. Chance — using the spectroscopic method — 

 determines the sum of both free and bound pyridine nucleotide. 



When DPNH is assayed with a method comprehending bound as 

 well as free DPNH (by killing the cells with hot NaOH and subsequent 

 determination of DPNH by means of alcohol dehydrogenase and 

 acetaldehyde), then we, too, find an increase in DPNH immediately 

 after the addition of NH4+ to glucose-oxidizing yeast cells (Holzer, H., 

 and Witt, I., unpublished experiments). 



Chance: You did not monitor your intracellular pH? It is possible 

 that the effect could be reversed by the addition of acid. 



