Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers.
After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.
Visvikis
A
A
Centre du Médicament, URA CNRS 597 Nancy, France.
Goergen
J L
JL
Oster
T
T
Bagrel
D
D
Wellman
M
M
Marc
A
A
Engasser
J M
JM
Siest
G
G
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms / Q000201:enzymology*; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification* / Q000378:metabolism
D005723:gamma-Glutamyltransferase