During the funded year of this project, we have characterized a transporter involved in ATP-dependent primary active transport of glutathione (GSH)-conjugates in human erythrocyte membrane and designated it as dinitrophenyl S-glutathione (Dnp-SG) ATPase because the use of Dnp-SG as a model substrate. Subsequently we showed that Dnp-SG ATPase was ubiquitous in human cell plasma membranes and that in addition to GSH-conjugates it was also involved in the ATP-dependent transport of bilirubin-conjugates, leukotrienes, and structurally unrelated compounds such as doxorubicin and other substrates of P-glycoprotein, a well characterized ATP-dependent pump overexpresed in multidrug resistance cancer cells. These studies, for the first time demonstrated the presence of an extremely versatile transporter (distinct from P-glycoprotein) in human cells which could actively transport diverse group of xenobiotics, drugs, and their phase I and phase II metabolites. To establish a unifying theme for the mechanisms of transport of such structually diverse compounds by Dnp-SG ATPase, studies are proposed in this application for its structural and functional characterization. Dnp-SG ATPase will be purified from human erytocytes and other tissues by Dnp-SG affinity chromatography and immunoaffinity chromatography to determine its structural and functional properties. The amino acid sequences of the peptide fragments of Dnp-SG ATPase generated by CNBr cleavage and isolated by HPLC and/or in SDS gels followed by transblotting on P-PVDF membranes will be determined. These sequences will be used to design and synthesize nucleotide probes to clone and sequence the cDNA of Dnp-SG ATPase to deduce its primary structure. Recombinant Dnp-SG ATPase will be prepared by expressing it in E. coli and/or other suitable vectors to get sufficient protein for its structural and functional characterization. Antibodies against Dnp-SG ATPase will be usssl in the alternate approaches for cloning. Possible genomic heterdgereity at Dnp-SG ATPase locus will be investigated to examine the existence of other related transporters at this locus. The kinetics of the ATP hydrolyzing activity of Dnp-SG ATPase stimulated by GSH-conjugates of xenobiotics and toxic products of lipid peroxidation such as 4- hydroxynonenal (4-HNE), and the substrates of P-glycoprotein (e.g. doxorubicin, vincristine) will be studied. Also the kinetics and mechanisms of the ATP-dependent transport of these compounds in the inside out vesicles (IOVs) prepared from erythrocyte membranes and in reconstituted proteoliposomes with native recombinant Dnp-SG ATPase will be studied. We will test the hypothesis whether Dnp-SG ATPase is a mediator of doxorubicin transport and hence resistance of P glycoprotein negative, doxorubicin resistant small cell lung cancer cell lines developed by us from parental NCI H-69 cell line. Studies proposed in this project will define the role of Dnp-SG ATPase in the protection mechanisms against structurally diverse xenobiotics and toxic endobiotics (such as 4-HNE), and will test the hypothesis that Dnp-SG ATPase may be involved in the mechanisms of drug resistance of cancer cells, particularly those which do not express P glycoprotein.