title: Drosophila melanogaster glutamate-cysteine ligase activity is regulated by a modifier subunit with a mechanism of action similar to that of the mammalian form.
abstract: Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis. In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence of a modifier subunit in invertebrates has not been described to date. We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM). A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately in Escherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an approximately 140-kDa complex. DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo. Enzyme kinetic analyses showed that DmGCLC has a K(m) for glutamate of 2.88 mm, but when complexed with DmGCLM, the K(m) for glutamate is 0.45 mm. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (K(i) = 0.03 mm), whereas inhibition of the GCL complex was mixed (K(i) = 0.67 mm), suggesting allosteric effects. In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges. A further mechanism for control of D. melanogaster GCL was found to be induction of DmGCLC by tert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.
authors.affiliation: Biomedical Research Center, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Fraser
authors.forename: Robert D C
authors.initials: RD
authors.lastname: Saunders
authors.forename: Lesley I
authors.initials: LI
authors.lastname: McLellan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D002850:Chromatography, Gel; D016895:Culture Media, Serum-Free; D003538:Cystamine / Q000494:pharmacology; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020224:Expressed Sequence Tags; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D008969:Molecular Sequence Data; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000975:Antioxidants; D016895:Culture Media, Serum-Free; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D021122:Protein Subunits; D011994:Recombinant Proteins; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003538:Cystamine