CHARACTERIZATION OF GLUTATHIONE UPTAKE IN BROAD BEAN LEAF PROTOPLASTS

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protopla sts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conju gates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-m ethionine) and peptides (glycine-glycine, glycine-glycine-glycine) aff ected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an appare nt K-m of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, w ith an apparent K-m of 7 mu M and 3.7 mM. It is concluded that the pla sma membrane of leaf cells contains a specific transport system for gl utathione, which takes up GSSG and GS conjugates preferentially over G SH. Proton flux measurements and electrophysiological measurements ind icate that GSH and GSSG are taken up with proton symport. However, a d etailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.