The anaplerotic and gluconeogenetic metabolism of baker's yeast was studied at the enzymatic level during glucose-ethanol diauxic growth in the presence and absence of aspartate. Of the two possible anaplerotic systems, only the pyruvate carboxylase by-pass was present during the whole growth process. The second system, the glyoxylate by-pass (isocitrate lyase as the indicator), like the specific enzymes of the gluconeogenetic metabolism, phosphoenolpyruvate carboxykinase and hexosediphosphatase began to appear only after the glucose had been consumed. The addition of glucose during the growth phase based on ethanol effected a rapid disappearance of phosphoenolpyruvate carboxykinase and hexosediphosphatase activities. The activity of pyruvate carboxylase decreased when the growth medium was supplied with asparate. The presence of aspartate had no effect on the activities of the other enzymes studied.
The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min. On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.
Fixation of C 0 2 by Saccharomyces cerevisiae growing under 14C02 in a chemostat was investigated. Under anaerobic conditions, CO, provided 6-5 & 1 % of the total carbon of yeast grown on glucose and 1.6 yo of the total carbon of yeast grown on glucose plus excess aspartate. Under aerobic conditions, 2-6 yo of the yeast carbon was derived from exogenous COB during growth on glucose or glycerol, and 3.3 yo during growth on pyruvate or ethanol. The distribution of the fixed carbon among chemical components of the yeast, including some individual amino acids, was determined. Equilibration of C 0 2 across the cell membrane was probably not quite complete. Under anaerobic conditions, the similar molar radioactivities of aspartate and glutamate indicated that oxaloacetate was not metabolically compartmented. The unequal labelling of aspartate and glutamate during aerobic growth was consistent with operation of the glyoxylate bypass and/or compartmentation of oxaloacetate. Increased C 0 2 fixation and labelling of carbohydrate during growth on pyruvate or ethanol are ascribed to the activity of phosphoezzolpyruvate carboxykinase. METHODS Organism and growth conditions. A baker's yeast, Saccharomyces cerevisiae strain no. 306 from the collection of our laboratories, was used. The yeast was cultivated continuously at a maintained pH of 4.5, 28 "C and a dilution rate of 0.1 h-l in a 2.7 1 Biotec fermenter, type FL103 (LKB, Sweden) with the carbon source as the growth-limiting factor. Except for some special arrangements described below, the
1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.
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