Fraser, Jennifer A.; Saunders, Robert D.C. and McLellan, Lesley I.
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|DOI (Digital Object Identifier) Link:||http://dx.doi.org/10.1074/jbc.M106683200|
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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 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 Km for glutamate of 2.88 mM, but when complexed with DmGCLM, the Km for glutamate is 0.45 mM. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (Ki 0.03 mM), whereas inhibition of the GCL complex was mixed (Ki 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.
|Item Type:||Journal Article|
|Extra Information:||Copyright held by the American Society for Biochemistry and Molecular Biology.
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|Academic Unit/Department:||Science > Life, Health and Chemical Sciences|
|Depositing User:||Robert Saunders|
|Date Deposited:||04 Jul 2006|
|Last Modified:||04 Dec 2010 21:01|
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