Molecular and functional characterization of recombinant human gamma-glutamyltransferase. Coupling of its activity to glutathione levels in V79 cells.
We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.
Thioudellet
C
C
Centre du Médicament, URA CNRS 597, Nancy, France.
Oster
T
T
Wellman
M
M
Siest
G
G
D000595:Amino Acid Sequence; D001881:Borates / Q000494:pharmacology; D002384:Catalysis; D002455:Cell Division; D002460:Cell Line; D002942:Circular Dichroism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D017997:Leukotriene C4 / Q000378:metabolism; D017998:Leukotriene D4 / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001881:Borates; D007555:Isoxazoles; D011994:Recombinant Proteins; D017997:Leukotriene C4; D012694:Serine; D017998:Leukotriene D4; D005723:gamma-Glutamyltransferase; D005978:Glutathione; C006303:acivicin