Cxcv. Phosphorylation in Living Yeast

EULER & ADLER [1935] recently described the preparation from yeast of a cell-free enzyme, "heterophosphatese" which catalyses the transference of phosphate from (muscle) adenosinetriphosphate to glucose or fructose with the formation of a hexosephosphate. Later Meyerhof [1935] showed that preparations of hexokinase contain "heterophosphatese". The natural inference from these experiments is that adenosinetriphosphate may act as a phosphate carrier in alcoholic fermentations by living yeast, in a manner analogous to its role in muscle glycolysis [see Meyerhof & Kiessling, 1935]. This receives material support by the isolation of the triphosphate in crystalline form from the trichloroacetic acid extract of fresh yeast by Wagner-Jauregg [1936] who concluded that it was identical with that of muscle. The amount obtained (60 mg. from 100 g. yeast) contains approximately 2% of the total phosphorus of the yeast. It seemed possible that the amount of adenosinetriphosphate present in yeast might be increased during the fermentation of sugar by synthesis from adenylic acid, and this question was investigated. Judging by the analysis of the sparingly soluble barium salts no increase in the triphosphate content was found if the yeast were extracted with trichloroacetic acid whilst fermenting sugar rapidly, but it was noticed that such extracts contained less orthophosphate than similar extracts from resting yeast. It is very generally inferred that alcoholic fermentation by living yeast involves esterification of orthophosphoric acid with carbohydrate, similar to that observed in cell-free fermentations, though there is no direct evidence in support of this view. The decrease in orthophosphate has therefore been investigated more fully, and it has been established that at the onset of fermentation of sugar by living yeast the acid-soluble orthophosphate rapidly decreases to a fairly constant level and rises again to the original level when the sugar is consumed. Whilst this work was in progress Lewitow [1936] showed that as a result of the fermentation of sugar both the (acid-soluble) orthoand pyro-phosphate contents of living yeast were decreased and came to the conclusion that pyrophosphate acts as a phosphate carrier in alcoholic fermentation in the same manner as adenosinetriphosphate in the glycolysis of animal cells. His experimental findings are open to criticism on several points. The routine deproteinization with trichloroacetic acid was preceded by 5 minutes' boiling of the yeast suspension, which materially alters the phosphorus distribution (see Table IV). The value for pyrophosphate was calculated from the hydrolysis curve in N HCI at 100° according to the equation