Glutamate-cysteine ligase (GCL) has a key influence on glutathione homeostasis. It has been proposed that mammalian GCL is regulated by the redox environment, and we show here that cysteine residues in the Drosophila melanogaster GCL modifier subunit (DmGCLM) can form covalent interactions with the catalytic subunit (DmGCLC) and modify its activity. Candidate components of intersubunit disulfides (Cys 213 , Cys 214 , and Cys 267 ) were identified using matrix-assisted laser desorption ionization time-of-flight spectroscopy of iodoacetamide-modified DmGCLM as well as examination of the evolutionary conservation of cysteines. Mutation of the 3 cysteine residues allowed DmGCLM to associate with DmGCLC, but inhibited the formation of intersubunit disulfides. This caused a 2-fold reduction in the catalytic efficiency of Drosophila GCL, although activity remained significantly higher than the catalytic subunit alone. The cysteine mutant was also more sensitive to inhibition by glutathione than the unmodified holoenzyme. Notably, human GCLM could substitute for DmGCLM in modification of DmGCLC activity. The role of DmGCLM in vivo was examined by analysis of a Drosophila mutant (l(3)L0580) containing a P-element insertion in Gclm. We found that the P-element is not responsible for the lethal phenotype and separated the recessive lethal mutation from the P-element by recombination. This yielded two fully viable and fertile recombinants bearing the P-element insertion, which Western and Northern blotting indicated is a severely hypomorphic allele of Gclm. Glutathione levels were ϳ2-fold lower in the Gclm L0580 mutants than in control strains, demonstrating the importance of DmGCLM in the regulation of glutathione homeostasis in vivo.The intracellular redox environment is of critical importance in cell physiology. It has a major influence on signaling pathways and cell fate in response to stress (1). Glutathione is one of the key influences on the redox state of the cell; and accordingly, intracellular glutathione levels are subject to multilateral regulatory mechanisms (2, 3).A major player in the regulation of glutathione homeostasis is glutamate-cysteine ligase (GCL), 1 which catalyzes the first and rate-limiting step in de novo synthesis of glutathione from precursor amino acids (4). GCL is located in the cytoplasm, and its activity appears to be regulated at several levels by the antioxidant status of the cell. In addition to being subject to transcriptional activation by pro-oxidants (5-7), evidence suggests that the catalytic activity is subject to redox regulation by the reversible formation of disulfide bridges between its two subunits (8, 9). Furthermore, GCL is subject to feedback inhibition by glutathione at concentrations that are physiologically relevant (10). The complex regulation of GCL activity highlights its pivotal role in controlling cellular glutathione synthesis.GCL is a heterodimer composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). The presence of GCLM modulates the catalytic prop...

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Much effort is focussed on understanding the structural and functional changes in the heart that underlie age-dependent deterioration of cardiac performance. Longitudinal studies, using aged animals, have pinpointed changes occurring to the contractile myocytes within the heart. However, whilst longitudinal studies are important, other experimental approaches are being advanced that can recapitulate the phenotypic changes seen during ageing. This study investigated the induction of an ageing cardiomyocyte phenotypic change by incubation of cells with hydroxyurea for several days ex vivo. Hydroxyurea incubation has been demonstrated to phenocopy age- and senescence-induced changes in neurons, but its utility for ageing studies with cardiac cells has not been examined. Incubation of neonatal rat ventricular myocytes with hydroxyurea for up to 7 days replicated specific aspects of cardiac ageing including reduced systolic calcium responses, increased alternans and a lesser ability of the cells to follow electrical pacing. Additional functional and structural changes were observed within the myocytes that pointed to ageing-like remodelling, including lipofuscin granule accumulation, reduced mitochondrial membrane potential, increased production of reactive oxygen species, and altered ultrastructure, such as mitochondria with disrupted cristae and disorganised myofibres. These data highlight the utility of alternative approaches for exploring cellular ageing whilst avoiding the costs and co-morbid factors that can affect longitudinal studies.

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