Differential regulation of glutamate‐cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation

Abstract

Glutamate-cysteine ligase (GCL) is a heterodimer of a catalytic subunit (GCLC) that possesses all of the enzymatic activity and a modifier subunit (GCLM) that alters the Ki of GCLC for GSH. We hypothesized that the expression of GCLM and the association of GCLM with GCLC was responsible for the apparent increase in GCL activity state observed in liver of rats fed low protein diets or in hepatocytes cultured in low-sulfur amino acid-containing medium. Therefore, we conducted a series of studies with rats and with a human hepatoma cell line (HepG2/C3A) to assess the role of GCLM and holoenzyme formation in the regulation of GCL activity in response to sulfur amino acid intake or availability. Increases in GCL activity in rat liver, as well as in HepG2 cells, were due to the additive effects of changes in GCLC amount and GCL kcat. The increase in GCL kcat was associated with increased holoenzyme formation, which was associated with an increase in the molar ratio of GCLM to GCLC. Furthermore, our results indicate that the GCLM level in rat liver is always limiting and that up-regulation of the GCLM level results in increased holoenzyme formation and an increase in kcat. This is the first report demonstrating that the catalytic efficiency of rat GCL is increased by holoenzyme formation and the first demonstration of differential up-regulation of the GCL subunits in response to cysteine deprivation.