Molecular and functional characterization of recombinant human γ‐glutamyltransferase

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human y-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 a-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. 7-Glutamyltransferase (GGT) is a cell surface enzyme involved in the metabolism of reduced (GSH) and oxidized (GSSG) glutathione and its S-substituted derivatives by initiating their breakdown. Nowadays its role in important biochemical pathways, such as detoxification or inflammatory responses, by processing GSH-conjugated electrophiles [ 11 and leukotrienes [2], is well established. Current interest for this enzyme is focused on its heightened activity in numerous