The transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) into inside-out vesicles from L1210 cells was employed to identify and characterize ATP-dependent efflux routes for DNP-SG. Measurements of ATP-dependent uptake at varying concentrations of [H]DNP-SG revealed the presence of two distinct transport systems. Transport at low substrate concentrations occurred predominantly via a high affinity system (K = 0.63 µM), whereas a low affinity system (K = 450 µM) predominated at high concentrations of substrate. The high affinity system was characterized by a potent inhibition by the glutathione conjugates of bromosulfophthalein (K = 0.09 µM) and ethacrynic acid (K = 0.44 µM), leukotriene C (K = 0.20 µM), and the taurate diconjugate of bilirubin (K = 0.10 µM). The low affinity transport system for DNP-SG exhibited a high affinity for bilirubin ditaurate (K = 1.8 µM), indoprofen (K = 3.0 µM), and biphenylacetic acid (K = 5.9 µM). Different results were obtained with an L1210/C7 variant which has a defect in the efflux of methotrexate and cholate. Vesicles from the latter cells contain the same low affinity transport activity as parental cells, but the high affinity route is absent and has been replaced by a system with an intermediate affinity for DNP-SG (K = 4.5 µM). These results indicate that L1210 cells contain two unidirectional efflux pumps for DNP-SG with substantial differences in inhibitor sensitivity. The high affinity system shows a binding preference for glutathione conjugates but can also accommodate large anionic conjugates, whereas the low affinity system has a binding preference for large organic anions. Results with the variant cells support the hypothesis that the high affinity transport system for DNP-SG also mediates the unidirectional efflux of methotrexate and cholate in intact L1210 cells.

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