Open Access2‐Deoxy‐ d ‐galactose Metabolism in Ascites Hepatoma Cells Results in Phosphate Trapping and Glycolysis Inhibition
Author(s) -
SMITH David F.,
KEPPLER Dietrich O. R.
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11293.x
Subject(s) - galactose , glycolysis , chemistry , metabolism , intracellular , biochemistry , phosphofructokinase , ethanol , pi
The metabolism of 2‐deoxy‐ d ‐galactose has been studied in AS‐30D rat ascites hepatoma cells in suspension. Using 2‐deoxy‐ d ‐[1‐ 14 C]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 P i in the form of 2‐deoxy‐ d ‐galactose 1‐phosphate resulted in a depression of free intracellular P i in spite of a concomitant increase in net 32 P i 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 P i trapping by removal of 2‐deoxy‐ d ‐galactose from the medium reversed the depressions of P i 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 P i 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.