2-Deoxy-d-galactose Metabolism in Ascites Hepatoma Cells Results in Phosphate Trapping and Glycolysis Inhibition

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Abstract
The metabolism of 2-deoxy-d-galactose has been studied in AS-30D rat ascites hepatoma cells in suspension. Using 2-deoxy-d-[1-14C]galactose and an alkaline ethanol deproteinization procedure, the quantitatively identified metabolites included 2-deoxy-d-galactose 1-phosphate comprising 99.3%, and UDP-2-deoxy-d-galactose and UDP-2-deoxy-d-glucose, together amounting to 0.4% of the total metabolites. After incubation for 5 h in the presence of 2-deoxy-d-galactose (1 mmol/l), the content of 2-deoxy-d-galactose 1-phosphate reached 35 mmol × (kg cells)−1. The rate of phosphorylation of 2-deoxy-d-galactose was rapid during the first 30 min and decreased to approximately 20% of this rate during the subsequent hours. The rapid trapping of Pi in the form of 2-deoxy-d-galactose 1-phosphate resulted in a depression of free intracellular Pi in spite of a concomitant increase in net 32Pi uptake from the medium and a decrease of ATP and other 5′-nucleotides. The rates of glucose utilization and lactate production were depressed by more than 80% in the presence of 2-deoxy-d-galactose (1 mmol/l). Interruption of Pi trapping by removal of 2-deoxy-d-galactose from the medium reversed the depressions of Pi and ATP and resulted in a rapid but incomplete relief of glycolysis inhibition. Crossover analysis of glycolytic intermediates indicated an inhibition at the 6-phos-phofructokinase step. The depression of glucose utilization may be mediated by the increased level of glucose 6-phosphate, a potent inhibitor of hexokinase. An additional inhibitory effect of a metabolite of 2-deoxy-d-galactose at the 6-phosphofructokinase step was indicated by crossover analysis after reversal of Pi and ATP depressions in the presence of a high intracellular content of 2-deoxy-d-galactose 1-phosphate. The quantitative analysis of the metabolites of 2-deoxy-d-galactose demonstrated the pre-dominance of the monophosphate and the negligible formation of UDP derivatives of this sugar analog in AS-30D hepatoma cells. This provides a system for the investigation of a galactose analog as a phosphate-trapping agent in the virtual absence of uridylate trapping.

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