γ glutamyl p nitroanilide

Determination of GGT activity was performed as described by Huseby and Srömme (9 (link)) using 22.5 mmol/l γ-glutamyl-p-nitroanilide (Sigma-Aldrich) as the donor and 123 mmol/l glycylglycine (Sigma-Aldrich) as the glutamate acceptor, with a volume fraction of 1:11. Confluent cell monolayers of human hepatoma HepG2 cells (American Type Culture Collection, Manassas, VA, USA) and hPLCs were pre-treated with GGsTOP for 2 h followed by further treatment with hypotonic lysis buffer (10 mM Tris-HCl, pH 7.8), and ground using a glass Dounce homogenizer (3432N75; Thomas Scientific, Swedesboro, NJ, USA) (30 strokes; 4°C). The cells were collected separately and amounts of p-nitroaniline formed were measured by spectrophotometry at 405 nm using a 722S Visible spectrophotometer (Shanghai Inesa Scientific Instrument, Shanghai, China). The units of enzyme activity were calculated using a molar extinction coefficient of 9.9 for p-nitroanilide. One unit of GGT activity was defined as the amount of enzyme required to release 1 μmol of substrate transformed/ml/min. The results were expressed as U/g cell protein. Protein content was measured using the bicinchoninic acid (BCA) method (Beyotime Institute of Biotechnology, Haimen, China).