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Abstract
The active outward translocation of phospholipid analogues from the inner to the outer membrane leaflet of human erythrocytes by the multi-drug resistance protein MRP1 (ABCC1) depends on intracellular reduced glutathione (GSH). Entrapment of ATP and increasing amounts of GSH inside resealed ghosts prepared from erythrocytes resulted in an up to six-fold increase of the translocation rate. Entrapped oxidized glutathione (GSSG) acted inhibitory but produced stimulation after addition of the disulphide-reducing reagent dithioerythritol. Modification of GSH by esterification of the C-terminal carboxylate of Gly, removal of the N-terminal Glu or substitution of the SH group by an anionic S-dicarboxyethyl or sulphonate group abolished stimulation. The effect of S-alkylation of GSH depended on the length of the alkyl group. S-methyl GSH was somewhat more effective than GSH, but maximal stimulation was similar. S-butyl GSH acted poorly stimulatory while S-hexyl GSH was essentially ineffective. Analyses of the kinetic data of translocation revealed Km values for GSH and methyl-GSH of respectively 7.4±2.4 and 4.9±1.1 mmol l−1. At high GSH levels and defined constant ATP levels using an ATP-regenerating system, the Km for ATP of the outward translocation was 0.16±0.02 mmol l−1. In the same system lacking GSH, the Km for ATP of the inward translocation by the aminophospholipid flippase was 0.53±0.23 mmol l−1.
ABC, ATP-binding cassette; GSH, glutathione; MRP, multidrug resistance protein; NBD-PC, 1-oleoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoyl]-sn-glycero-3-phosphocholine; NBD-PS, 1-oleoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl]-sn-glycero-3-phospho-L-serine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine
ABC, ATP-binding cassette; GSH, glutathione; MRP, multidrug resistance protein; NBD-PC, 1-oleoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoyl]-sn-glycero-3-phosphocholine; NBD-PS, 1-oleoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl]-sn-glycero-3-phospho-L-serine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine