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.
Highlights
Cellular injury from reactive oxygen species and electrophilic agents is inhibited by glutathione, an abundant and essential tripeptide thiol
Glutamate-cysteine ligase (GCL) activity seems to be regulated by cellular thiol antioxidant balance, and in mammals, this control mechanism is principally mediated by a non-catalytic polypeptide (GCLM) that dimerizes with the catalytic subunit of GCL (GCLC) [1, 2]
The apparent inhibition constant (Ki) values for glutathione were 0.03 mM for D. melanogaster GCLC (DmGCLC) and 0.67 mM for the DmGCL holoenzyme (Table I). These findings show that Drosophila melanogaster has a functional modifier subunit (DmGCLM) significantly reduces the sensitivity of the catalytic subunit to inhibition by glutathione, possibly by generating a conformational change preventing access of glutathione to the active site in a manner similar to the model proposed by Meister and co-workers [2]
Summary
Construction of DmGCL Constructs—The open reading frame (ORF) of the gcl gene was amplified from pDmGCS4.3.3 [10] by PCR using upstream (5Ј-GGGAATTCATATGGGTCTACTGAGCGAGGGC-3Ј) and downstream (5Ј-GCCTTAACTCGAGTCATTTCTCCTCGCAGCAGCC3Ј) oligonucleotides designed to insert EcoRI and NdeI sites at the 5Ј-end and an XhoI site at the 3Ј-end of the ORF. Bacterial cell pellets were resuspended in 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, 5 mM imidazole, and 0.1% (v/v) Igepal, and recombinant proteins were purified using nickel-agarose chromatography (QIAGEN Inc.) according to the manufacturer’s instructions. Gel Filtration Chromatography—Recombinant protein samples were dialyzed overnight against 3 liters of Buffer A (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM L-glutamate, and 5 mM MgCl2) containing 10 mM glutathione. For inhibition studies with L-buthionine-(SR)-sulfoximine (BSO), GCL samples were incubated at 25 °C with 1.5 M BSO in 100 mM Tris-HCl (pH 8.2), 20 mM MgCl2, and 5 mM ATP for 10 min prior to kinetic analyses. GCL inhibition studies with cystamine (2,2Ј-dithiobis(ethylamine)) were performed by incubating GCL samples with 1.5 M cystamine in 100 mM Tris-HCl (pH 8.2) for 10 min at 25 °C prior to kinetic analyses. Statistical Analyses—Statistical analyses were performed using Student’s paired t test
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