REGULATION OF GLUTATHIONE SYNTHESIS IN SUSPENSION-CULTURES OF PARSLEY AND TOBACCO

Experiments in vitro have shown that gamma-EC synthesis, the first step in GSH formation, is subject to feedback inhibition by physiological GSH concentrations. In order to evaluate the role of this feedback inhibition on gamma-EC synthetase in vivo GSH synthesis was modulated in suspension cultures of P. crispum and N. tabacum by administration of cadmium. The alterations in the thiol contents were measured and in addition the effect of Cd exposure on gamma-EC synthetase (E.C. 6.3.2.2) and GSH synthetase (E.C. 6.3.2.3) was studied. Decreasing cellular GSH concentrations by cadmium induced PC synthesis caused 7-10 fold increase in the rate of glutathione synthesis as measured by the accumulation of (gamma-EC)(n)G. This increase was not linked to an increase in extractable activities of gamma-EC- or GSH synthetase in parsley. In tobacco the activities of gamma-EC- and GSH synthetase increased by a factor of 1.6 and 1.8, respectively, after 3 d of Cd exposure. In both species the exposure to Cd resulted in an increased cellular gamma-EC content that reached a plateau within 24 h, and in a doubling of the cysteine content. In vitro experiments showed that GSH synthetase activity is inhibited by cadmium concentrations that have no effect on gamma-EC synthetase activity. This may explain the accumulation of gamma-EC in Cd exposed cells. Incubation with 0.25 mM cysteine did not effect the gamma-EC- and GSH content in tobacco cells. In parsley the cellular GSH content increased threefold and the gamma-EC content twofold and stayed constant thereafter at the elevated levels. Taken together the results show that GSH synthesis in vivo is controlled by feedback inhibition as well as by the supply with cysteine. In the latter case the feedback inhibition may act as a kind of safety valve and prevent the accumulation of unphysiological GSH concentrations if the supply of cysteine is too large.