We investigated whether hepatocytes permeabilized with α-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2,6-bisphosphate [Fru(2,6)P2], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P2, rates of detritiation of [2-3H]glucose and [3-3H]glucose were similar. Exogenous Glc6P (1mM) and to a lesser extent fructose 6-phosphate, but not Fru(1,6)P2, decreased the rate of detritiation of [3-3H]glucose. During incubation with 25mM glucose and Glc6P (0.2Ő1mM), with either [3-3H]glucose or [3-3H]Glc6P as labelled substrate, there was dilution of metabolism of [3-3H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P2. They provide an alternative explanation for the concept that there is more than one Glc6P pool.
Use of α-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes
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Marta CASCANTE, Josep J. CENTELLES, Loranne AGIUS; Use of α-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes. Biochem J 15 December 2000; 352 (3): 899–905. doi: https://doi.org/10.1042/bj3520899
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