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

Despite the central role of gamma-glutamylcysteine synthetase (gamma GCS) i n lung antioxidant defenses, the limited studies of the activity of this en zyme in respiratory cells have produced variable results. This study has ex amined the factors, which may influence the measurement: of gamma GCS activ ity in cultured human lung epithelial cells (A549). Although a source of po tential error, gamma GCS activity In A549 cell extracts did not vary signif icantly when appropriately assayed by three different methods or after remo val of the endogenous inhibitor, glutathione (GSH). However, gamma GCS acti vity did increase significantly during the early stages of cell proliferati on (3.50 +/- 0.3 vs. 2.35 +/- 0.16 nmol/min/10(6) 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 alter ed gamma GCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells , at 0.25 x 10(5) and 0.58 x 10(5) cells/cm(2), respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm(2), respectively, p < .001) during the earl y stages of cell proliferation. In addition, gamma GCS activity and GSH con tent were highest in A549 cells grown in medium containing cystine as the p redominant sulfur-containing amino acid. These results suggest that gamma G CS 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 investiga tors. Whether gamma GCS has an important role in the early phase of cell pr oliferation needs further investigation. (C) 1999 Elsevier Science Inc.