The catalytic activity of gamma-glutamylcysteine ligase (gamma-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes-Woolf analyses ([S]/nu vs [S]) indicated that gamma-GCL had significantly higher affinities for its substrates in the young than in the old flies. The K,, values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM, for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of gamma-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native gamma-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies. (C) 2004 Elsevier Inc. All rights reserved.