An unusual high-Km hexokinase is expressed in the mhAT3F hepatoma cell line.

In most hepatoma cells, the high-Km GLUT2/glucokinase proteins are replaced by the ubiquitous low-Km GLUT1/hexokinase type I proteins. In the mhAT3F hepatoma cells, the stimulatory effect of glucose on gene expression and glycogen accumulation was not maximal at 5 mmol/liter glucose. This response to high glucose is observed in mhAT3F cells, where GLUT2 was expressed, but not glucokinase (assessed by Northern blotting and reverse transcription-polymerase chain reaction). A low-Km hexokinase activity (19.6 +/- 3.8 milliunits/mg of protein) was present, but a high-Km (40 mmol/liter) hexokinase activity (13.9 +/- 2.5 milliunits/mg) was also detected in mhAT3F cells. The high-Km hexokinase activity was dependent on both ATP (or PPi) and glucose in the assay and was recovered in a 10-50-kDa fraction after filtration. A 30-kDa protein was detected using an anti-glucokinase antibody and localized by confocal microscopy at the same sites as glucokinase in hepatocytes. In FAO cells, the high-Km hexokinase activity and 30-kDa protein were not found. We conclude that a high-Km hexokinase activity is present in mhAT3F cells. This might explain why the effects of glucose on gene expression were not maximal at a glucose concentration of 5 mmol/liter. A 30-kDa protein identified using an anti-glucokinase antibody may be responsible for this activity present in mhAT3F cells.