Use of alpha-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes

We investigated whether hepatocytes permeabilized with a-toxin from Staphyl ococcus aureus are a valid model for studying the channelling of intermedia tes of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2,6-bisphosyhate [Fru(2,6)P-2], but maintain cell integrity i n the presence of ATP as judged by the retention of cytoplasmic enzymes. Du ring incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P-2 rates of detritiation of [2-H-3]glucose and [3-H-3]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1,6)P-2, decreased the rate of detritiati on of [3-H-3]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-H-3]glucose or [3-H-3]Glc6P as labelled substrate, the re was dilution of metabolism of [3-H-3]glucose with increasing Glc6P but n o overall increase in glycolytic flux from glucose and Glc6P, indicating th at glycolysis is apparently saturated with Glc6P despite the permeability o f the cells to this metabolite. These findings could be explained by partia l channelling of Glc6P between glucokinase and glycolysis in the presence o f saturating concentrations of Fru(2,6)P-2. They provide an alternative exp lanation for the concept that there is more than one Glc6P pool.