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

S. Ray, Neil Misso, Jason Lenzo, Cleo Robinson, Philip Thompson

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    16 Citations (Scopus)

    Abstract

    Despite the central role of gamma-glutamylcysteine synthetase (gamma GCS) 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 gamma GCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gamma GCS 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, gamma GCS activity did increase significantly during the early stages of cell proliferation (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 altered 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 early stages of cell proliferation. In addition, gamma GCS 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 gamma GCS 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 gamma GCS has an important role in the early phase of cell proliferation needs further investigation. (C) 1999 Elsevier Science Inc.
    Original languageEnglish
    Pages (from-to)1346-1356
    JournalFree Radical Biology and Medicine
    Volume27
    DOIs
    Publication statusPublished - Dec 1999

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