DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

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Glutathione (GSH) and the related enzyme systems such as GSH S- transferase (GST) play an important role in the protection of human tissues from toxic xenobiotics and endogenous oxidative stress. GST catalyzes the conjugation of a wide variety of toxic electrophilic xenobiotics to GSH. The GSH-xenobiotic conjugates are metabolized to mercapturic acids in most of the human tissues, but in erythrocytes, they cannot be metabolized further because all the enzymes of the mercapturic acid pathway are not present in this tissue. We have demonstrated that the conjugate of 1-chloro-2, 4-dini-trobenzene and GSH (S-dinitrophenyl glutathione, abbreviated and Dnp-SG) is actively transported out of the erythrocytes by a novel ATPase not related to any known ion pump ATPase and have now developed a procedure to isolate this Dnp-SG dependent ATPase using affinity chromatography over a column of Dnp-SG bound to CNBr activated sepharose 4B using lubrol as a detergent. This transporter protein will be isolated from erythrocyte membrane and characterized for its structural and kinetic properties. The kinetic parameters of the Dnp-SG ATPase will also be determined using conjugates of other xenobiotics in order to examine whether or not this system is a general mechanism for the transport of GSH-xenobiotic conjugates. The purified transporter protein will be reconstituted in proteoliposomes. The kinetics of transport in the liposomes as well as in the inside-out vesicles (IOVs) prepared from erythrocyte ghosts will be studied using 3H Dnp-SG. The interrelationship between Dnp-SG and oxidized glutathione (GSSG) transport systems will be studied by determining the effect of GSSG on Dnp-SG stimulated ATP hydrolysis and also by isolating the GSSG transporter using analogous procedure of GSSG-CNBr activated sepharose 4B affinity chromatography. The antibodies raised against erythrocyte Dnp-SG ATPase will be used for immunocytochemical localization of transport-protein in the erythrocytes and other tissues. In addition, we will investigate the structural, kinetic, and non-catalytic binding properties of the novel GST isoenzyme (GST sigma) recently described by us in erythrocytes. Because this isoenzyme has high activity towards epoxide substrates and is highly hydrophobic, we believe that it is a membrane associated enzyme and may be important in defense against lipid peroxidation. The proposed studies on Dnp-SG ATPase and GST sigma will significantly enhance our understanding of the detoxification mechanisms in erythrocytes.