290 Discussion 



is postulated to be rate-limiting in this process and one must invoke a 

 hypothesis of localization of DPN as well as that already presented 

 earlier on ATP localization (Lynen, F., and Koenigsberger, R. (1951). 

 Justus Liebigs Ann. Chem., 573, 60). (See also p. 256). 



Backer : Was the DPN measured by the absorption given by the 

 triose phosphate dehydrogenase-DPN compound? 



Chance: The DPN assay of Fig. lA was based on the assumption that 

 the starved cell contained nearly all its pyridine nucleotide in the oxi- 

 dized form. In other experiments the DPN is measured by the amount 

 of complex (Racker, E., and Krimsky, I (1952). J. biol. Chem., 198, 

 731 ; Chance, B. (1954). In The Mechanism of Enzyme Action, p. 444, 

 Ed. McElroy, W. D., and Glass, B. Baltimore: Johns Hopkins Press) 

 that disappears upon the addition of iodoacetate to the starved yeast 

 cell. 



Racker: What happens when DPNH disappears? Does acetaldehyde 

 act as acceptor? 



Chance: The rapid oxidation of pyridine nucleotide following its 

 initial reduction upon glucose addition is probably due to a-glycerol- 

 phosphate dehydrogenase activity ; addition of glucose to cells pretreated 

 with iodoacetate and ethanol causes a rapid oxidation of reduced 

 pyridine nucleotide. While it is possible that a system other than 

 a-glycerolphosphate dehydrogenase could be responsible, none is known 

 that could act under these conditions. In the absence of iodoacetate, 

 acetaldehyde accumulation could also contribute to this oxidation 

 reaction and Prof. Holzer's assays for acetaldehyde accumulation in the 

 first 30 seconds after glucose addition also favour this view. 



Now returning to Dr. Backer's question on ammonia and to Prof. 

 Holzer's proposed explanation for the increase of aerobic fermentation 

 caused by the addition of NH4+, it is worth while to consider not only 

 the effect of NH4+ on the a-ketoglutarate equilibrium but also the 

 steady state concentration of pyridine nucleotide. It is known that 

 ammonia and amines penetrate the yeast cell and cause changes of 

 intracellular pH which can be studied with suitable indicators (Brand, 

 K. M. (1945). Acta physiol. scand., 10, suppl. 30). In fact, we have 

 attempted to compute the equilibrium constant for the following 

 chemical reaction on the basis of titrations of intracellular pyridine 

 nucleotide with acetaldehyde and alcohol. 



H+ + DPNH + acetaldehyde = DPN+ + ethanol 



Fig. 2A provides a verification of the shift of the steady state of 

 reduced pyridine nucleotide towards reduction by increasing concen- 

 trations of ammonia. In this particular experiment the yeast cells were 

 suspended in an alkaline phosphate medium of pH 9 • 3 in order to pro- 

 duce an optimal effect of the ammonia. In addition, the oxidation- 

 reduction state of the pyridine nucleotide was set at a relatively oxidized 

 level by an alcohol-aldehyde "redox buffer" (23 mivi and 1-5 mM, 

 respectively). Aliquots of a solution of 1 • 5 m ammonia were added to 

 the anaerobic cells. The effectiveness of ammonia in producing increased 

 reduction of intracellular pyridine nucleotide is clearly demonstrated. 



