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

GCL (glutamate–cysteine ligase) is a heterodimer of a GCLC (GCL catalytic subunit) that possesses all of the enzymatic activity and a GCLM (GCL modifier subunit) that alters the K(i) of GCLC for GSH. We hypothesized that the expression of GCLM and the association of GCLM with GCLC were responsible for the apparent increase in GCL activity state observed in the liver of rats fed low-protein diets or in hepatocytes cultured in low-sulphur amino acid-containing medium. Therefore we conducted a series of studies using rats and a human hepatoma (HepG2/C3A) cell line to assess the role of GCLM and holoenzyme formation in the regulation of GCL activity in response to sulphur 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 the amount of GCLC and the k(cat) for GCLC. The increase in the k(cat) for GCLC 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 the k(cat). 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

Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

Cystathionine γ-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by λ genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice