MOLECULAR AND FUNCTIONAL-CHARACTERIZATION OF RECOMBINANT HUMAN GAMMA-GLUTAMYL-TRANSFERASE - COUPLING OF ITS ACTIVITY TO GLUTATHIONE LEVELS IN V79 CELLS

We previously described the establishment of a transfected cell line ( V79HGGT) that stably produces the highest recombinant human gamma-glut amyltransferase (GGT) activity. We now report the utilization of V79HG GT as a model system for studying human GGT The papain-solubilized rec ombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequen cing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native h uman enzymes. The circular dichroism analysis indicated an a-helical c ontent of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutat hione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell lin e, depending on, but differentially affected by, alteration of the cul ture medium. Elevated GGT activity was associated with a 2.5-fold redu ced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Pos sible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important fact or determining the steady-state content of GSH.