Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement.
Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) 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 gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS 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, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 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 altered gammaGCS 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/cm2, 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/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS 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 gammaGCS 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 gammaGCS has an important role in the early phase of cell proliferation needs further investigation.
Ray
S
S
Asthma and Allergy Research Institute, and Department of Medicine, The University of Western Australia, Perth, Australia.
Misso
N L
NL
Lenzo
J C
JC
Robinson
C
C
Thompson
P J
PJ
D002452:Cell Count; D002455:Cell Division; D002460:Cell Line; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D013997:Time Factors
D003470:Culture Media; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione