ATP-Dependent Transport of Glutathione-N-Ethylmaleimide Conjugate across Erythrocyte Membrane

Abstract

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-ethylmaleimide-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°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 K_{m 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 K_m for NEM-SG was 5.6 ± 1.51 μM with a V_max of 7.30 ± 0.69 nmol/mg protein/h and the high K_m for NEM-SG was 1.35 ± 0.14 mM with a V_max of 65.1 ± 3.5 nmol/mg protein h. With respect to ATP, the NEM-SG transport had a low K_m of 0.12 ± 0.004 mM and a high K_m 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 K_m transport. GSH stimulated the low K_m transport 1.7-fold. Both low and high K_m components of NEM-SG transport significantly declined when ATP was substituted with CTP, UTP, or GTP. GSSG and Dnp-SG competitively inhibited the low K_m NEM-SG transport (K_i = 18.5 ± 2.9 and 1.32 ± 0.16 μM, respectively) whereas the high K_m 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 K_m mechanisms, the latter being shared by GSSG.