Visvikis A, Goergen J L, Oster T, Bagrel D, Wellman M, Marc A, Engasser J M, Siest G
Centre du Médicament, URA CNRS 597 Nancy, France.
Clin Chim Acta. 1990 Nov 5;191(3):221-32. doi: 10.1016/0009-8981(90)90023-l.
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.
在筛选了不同的人肝癌细胞系后，我们观察到HepG2和PLC/PRF/5细胞系均能自然产生大量γ-谷氨酰转移酶。我们优化了HepG2细胞的培养条件，观察到当细胞在微载体上培养时可获得更高的细胞密度，尤其是使用Cytodex 3微载体时；并且当基础培养基DMEM添加5%胎牛血清和6 mmol/L谷氨酰胺时细胞生长最佳。这些培养条件使我们在培养约150小时后能够产生最高量的γ-谷氨酰转移酶（GGT）。从HepG2细胞获得的GGT经过部分纯化，并对其一些理化性质进行了表征。连续的伴刀豆球蛋白A凝胶色谱将活性分离为两个峰，这表明来自HepG2的GGT糖基化并不均匀。木瓜蛋白酶处理的HepG2 GGT在SDS-PAGE中显示分子量约为120 kDa，且以单链蛋白形式迁移。该GGT的免疫学和动力学性质与其他人GGT（肝脏、肾脏和血清来源）相似。看来HepG2 GGT可能是制备人酶参考物质的一个来源。
