Glutathione-linked pathways in drug resistance: Characterization and functional reconstitution of human erythrocyte glutathione-conjugate transporter

The glutathione-conjugate transporter, S-(2,4-Dinitrophenyl) glutathione (DNP-SG) ATPase described by us previously has been purified from human erythrocytes. DNP-SG ATPase showed a band at 38 kDa in SDS gels which was not recognized by the monoclonal antibodies against multidrug resistance associated protein (MRP). A saturable photoaffinity labeling of the 38 kDa band was observed with 8-azido ATP (K(d) = 2 μM). The transporter catalyzed ATP hydrolysis which was stimulated in the presence of glutathione-conjugate of 1-chloro-2,4-dinitro benzene (DNP-SG) as well as the cationic amphiphilic chemotherapeutic drug, doxorubicin (DOX). When reconstituted in artificial liposomes, DNP-SG ATPase mediated ATP-dependent, saturable transport of DOX (K(m) 2.4 μM, V(max) 194 nmol/min/mg) as well as DNP-SG (K(m) 36 μM, V(max) 433 nmol/min/mg). The K(m) for ATP for both substrates was about 2.5 mM. Transport of DOX was competitively inhibited by DNP-SG and vice versa.