Poly(gamma-glutamylcysteinyl)glycine synthesis in Datura innoxia and binding with cadmium. Role in cadmium tolerance

The effects of Cd on poly(gamma-glutamylcysteinyl)glycine [(gammaEC)nG] biosynthesis and formation of (gammaEC)nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl2 was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (gammaEC)nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl2. However, rates of (gammaEC)nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with L-[35S]cysteine resulted in equivalent rates of incorporation into the (gammaEC)nG of both cell lines during the initial 24 hoursafter Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (gammaEC)nG synthesis in sensitive cells is operational and that relative overproduction of (gammaEC)nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (gammaEC)nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.