Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells:: Factors influencing its measurement

Despite the central role of γ-glutamylcysteine synthetase (γGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of γGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, γGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, γGCS activity did increase significantly during the early stages of cell proliferation (3.50 ± 0.31 vs. 2.35 ± 0.16 nmol/min/106 cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered γGCS activity (3.11 ± 0.14 vs. 4.04 ± 0.50 nmol/min/106 cells, at 0.25 × 105 and 0.58 × 105 cells/cm2, respectively, p < .001) and GSH content (45.43 ± 4.43 vs. 63.64 ± 3.28 nmol/106 cells at 0.25 × 105 and 0.58 × 105 cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, γGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that γGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether γGCS has an important role in the early phase of cell proliferation needs further investigation.