Erythrocytes detoxify toxic electrophilic xenobiotics and endogenously generated products of lipid peroxidation by conjugating then to glutathione (GSH), through a reaction catalyzed by glutathione S-transferases. In humans these GSH-conjugates are effluxed through a primary active transport mechanism mediated by a different ATPase of erythrocyte membranes which have been designated as S-dinitrophenyl glutathione ATPase (Dnp-SG ATPase) because of its ability to stimulate ATP hydrolysis in the presence of its substrate, S-dinitrophenyl glutathione (Dnp-SG) in a cell free system. In this continuation proposal, further structural and functional characterization of Dnp-SG ATPase is proposed. Dnp-SG ATPase purified through affinity chromatography will be analyzed for its amino acid composition, N-terminal sequence(s), subunit composition, and molecular weight. Kinetic properties of Dnp-SG ATPase including in vitro steady state kinetics of uptake of the substrate (Dnp-SG) by erythrocyte membrane inside out vesicles (IOVs), as well as by reconstituted proteoliposomes incorporated with purified Dnp-SG ATPase will be studied. In order to elucidate the physiological role of DNP-SG ATPase in the protection of erthyrocyte membrane, the transport of GSH-conjugates of electrophilic products of lipid peroxidation by erythrocytes will be studied in situ as well as in the IOVs. A mechanistic interrelationship between Dnp-SG ATPase and the P-glycoprotein mediated drug efflux pump, overexpressed in multi-drug resistant (MDR) cancer cell lines, has been recently suggested. This is supported by preliminary studies, which show that the transport of Dnp-SG from erthryocytes is inhibited by adriamycin, a model substrate for P-glycoprotein. The functional and structural interrelationship(s) between Dnp-SG ATPase transporter and the P-glycoprotein efflux pump will therefore, be studied using erthyrocytes and MDR cell lines. The kinetics of adriamycin dependent inhibition of the uptake of Dnp-SG by erthyrocyte membrane IOVs and the effect of Dnp-SG, and other Dnp-SG ATPase substrates on P-glycoprotein mediated efflux of adriamycin from LZ III N cells will be studied. The effect of P-glycoprotein substrates on Dnp-SG stimulated ATP hydrolysis by purified Dnp-SG ATPase will also be studied. Expression of Dnp-SG ATPase in other tissues as well as in tumor and MDR cell lines will be studied by Western blot analyses using the polyclonal antibodies against Dnp-SG ATPase raised by the investigators.
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