ATP-dependent transport of glutathione-N-ethylmaleimide conjugate across erythrocyte membrane.

Prevailing controversies regarding the identity and nature of S-(2,4-dinitrophenyl) glutathione (Dnp-SG) and GSSG transport system(s) led us to examine xenobiotic-SG transport from human erythrocytes and into inside-out vesicles (IOV) using N-ethyl-maleimide-glutathione conjugate (NEM-SG) as substrate. Efflux of NEM-SG from intact erythrocytes was linear over a period of 4 h, occurred against a concentration gradient, and required energy. No transport of NEM-SG was observed when endogenous ATP was exhausted by preincubation of the erythrocytes for 8 h at 37 degrees C in the absence of glucose. When cellular GSH was partially conjugated with NEM to form 1.5 and 1.0 mM NEM-SG, and the remaining GSH was oxidized with t-butylhydroperoxide to generate 0.2 and 0.4 mM GSSG, respectively, the extrusion of NEM-SG from erythrocytes was not inhibited. The kinetics of NEM-SG transport in intact erythrocytes were monophasic; the Km NEM-SG was 0.62 mM +/- 0.24. However, in IOV two components of NEM-SG transport with respect to NEM-SG and ATP were discernible. The low Km for NEM-SG was 5.6 +/- 1.51 microns with a Vmax of 7.30 +/- 0.69 nmol/mg protein/h and the high Km for NEM-SG was 1.35 +/- 0.14 mM with a Vmax of 65.1 +/- 3.5 nmol/mg protein h. With respect to ATP, the NEM-SG transport had a low Km of 0.12 +/- 0.004 mM and a high Km of 0.52 +/- 0.052 mM. Both components of NEM-SG transport were inhibited by fluoride, o-vanadate, p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid). However, NEM (1 mM) inhibited only the high Km transport. GSH stimulated the low Km transport 1.7-fold. Both low and high Km components of NEM-SG transport significantly declined when ATP was substituted with CTP, UTP, or GTP. GSSG and Dnp-SG competitively inhibited the low Km NEM-SG transport (Ki = 18.5 +/- 2.9 and 1.32 +/- 0.16 microns, respectively) whereas the high Km transport was inhibited by Dnp-SG but not by GSSG. These findings suggest that glutathione S-conjugates may be transported out of erythrocytes by both the high and the low Km mechanisms, the latter being shared by GSSG.