Oster T, Visvikis A, Thioudellet C, Fournel-Gigleux S, Wellman M, Siest G
Centre du Médicament, URA CNRS 597, Nancy, France.
Toxicology. 1993 Oct 5;82(1-3):151-67. doi: 10.1016/0300-483x(93)02609-k.
gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
γ-谷氨酰转移酶（GGT）是一种参与谷胱甘肽代谢的酶，其在血清和器官中的活性变化是肿瘤发生前期过程、酒精滥用及药物诱导的重要标志物。为阐明GGT在各种代谢途径中的作用，我们构建了一个稳定转染的V79细胞系，该细胞系可高效表达人GGT。将编码人肝癌Hep G2 GGT的全长cDNA亚克隆至受SV40早期启动子调控的表达载体中，并用于转染V79细胞。筛选出一个细胞系，其GGT活性为每毫克蛋白质2个单位，这是迄今为止报道的最高水平之一。从该细胞系中纯化得到的重组GGT呈现出预期的异二聚体结构，两个亚基以唾液酸化和不同糖基化的异构体形式存在，平均分子量分别为80 kDa和29 kDa。其催化特性与人血清和Hep G2 GGT的催化特性相同。因此，新构建的细胞系可用于生产人GGT，并有望作为药理学研究的替代模型。
