268 F. Lynen, et al. 



the blocked oxidative phosphorylation. In the first period, 

 therefore, dephosphorylation exceeds phosphorylation. For 

 our discussion it is important that glucose-6-phosphate 

 changes in parallel with ATP, and drops rapidly after respira- 

 tion has been blocked. This leaves no doubt that, in yeast, 

 mitochondrial ATP can be used for the initial phosphorylation 

 of sugar. 



The behaviour of fructose-1 : 6-diphosphate is quite un- 

 expected: no change takes place immediately after cyanide 

 poisoning; after 10 seconds, the fructose — 1 : 6-diphosphate 

 begins to rise until after about 2 minutes it reaches the higher 

 level which corresponds to the anaerobic steady-state value. 

 There are three possible explanations for this experimental 

 result, which has been confirmed many times. Firstly, phos- 

 phofructokinase might have a much higher affinity for ATP 

 than hexokinase. Lowering the ATP level, then, could have 

 only little influence on the rate of fructose diphosphate 

 formation. This theory was ruled out when Bryant (1958, 

 unpublished), working in our laboratory, determined the 

 Michaelis constant of the yeast enzyme, and found it to be 

 close to the Michaelis constant of yeast hexokinase. Secondly, 

 the rate of the ATP-dependent formation of fructose diphos- 

 phate might decrease simultaneously with the ATP level. At 

 the same time, however, this decreased formation could be 

 compensated by the diminished fructose diphosphate de- 

 gradation. For instance, lack of DPN, following cyanide 

 poisoning of respiration, would inhibit its further oxidation 

 via triose phosphate. In fact, Chance — by means of his 

 spectrophotometric method (Chance and Hess, 1956) — found 

 that in the initial period after cyanide addition DPN is 

 almost completely reduced. It takes 2-3 minutes before the 

 stationary DPN/DPNH ratio of fermentation is reached 

 (Fig. 9). However, it is possible that this fast DPNH formation 

 may actually be due to triose phosphate dehydrogenation 

 which, by reason of the cell being flooded with inorganic 

 phosphate, is out of control for a short while. Experiments 

 with DNP show that depletion of oxidized DPN cannot 



