Pyruvic acid, which is known to be an intermediate of glucose fermentation, was added to yeast during adaptation to galactose fermentation. It was found to neutralize the inhibition by sodium fluoride, and to decrease the apparent time of adaptation from 90 to about 45 or 60 minutes. In control experiments, it was shown that intact yeast is unable appreciably to ferment or decarboxylate alone, although it oxidizes the compound readily.
Experiments in which galactose and pyruvate were added at various times and in different orders were used to eliminate the possible complications of the rates at which these compounds penetrate the cells. Under these conditions, it was not possible to reduce the time of adaptation below 45 minutes. It was concluded that the rôle of added pyruvate was to serve as a source of acetaldehyde, which in turn could accept hydrogen and be reduced to alcohol. Substances, such as triose phosphate, which could serve as hydrogen donors were not produced from galactose in appreciable quantities until 45 minutes had elapsed. This time was therefore inferred to be the true adaptation time, during which the first synthesis of adaptive enzymes occurred.
Some determinations of the distribution of phosphorylated intermediates at various stages during the adaptive process were carried out. It was found that ATP, which usually serves to phosphorylate hexoses, accumulates during the preadaptive phase, diminishes rapidly after 60 minutes, and subsequently increases once more. The source of the ATP phosphate appeared to be PPA or triose phosphate initially present in the cells. It was inferred that the adaptive enzyme was concerned with the phosphorylation of galactose and the conversion of the phosphate ester to a glucose ester, which could then be fermented by the normal enzymes of the cell.
Added ATP was found to stimulate adaptation to a considerable extent, but did not shorten the time of adaptation below 75 minutes. This seemed consistent with the rôle of ATP as a phosphate donor for galactose. Creatine was found to inhibit adaptation to some degree, in agreement with its known ability to act as a competitive phosphate acceptor.
It was demonstrated that yeast produces, during and after adaptation, substances which shorten the apparent adaptation time of fresh samples of yeast. In agreement with our other findings, it appeared that such substances were not formed before about 45 minutes. They are probably not metabolic intermediates, and may be identical with the adaptive principle which can be extracted from adapted cells.