Functional reconstitution of RLIP76 catalyzing ATP-dependent transport of glutathione-conjugates.
RLIP76, a stress-responsive, multi-functional protein with multi-specific transport activity towards glutathione-conjugates (GS-E) and chemotherapeutic agents is frequently overexpressed in malignant cells. Our recent studies suggest that it plays a prominent anti-apoptotic role selectively in cancer cells. The present studies were performed to compare RLIP76 activity towards glutathione-conjugates in recombinant and K562 human erythroleukemia cells. The purity and identity of recombinant and K562 RLIP76 was established by SDS-PAGE and Western blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Comparison of ATPase activity and transport kinetics for DNP-SG and GS-HNE between recombinant vs. K562 RLIP76 revealed higher specific activity of ATPase and transport for recombinant purified RLIP76, indicating that additional factors present in recombinant purified RLIP76 can modulate its transport activity.
Singhal
Sharad S
SS
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107-2699, USA. ssinghal@hsc.unt.edu
Sehrawat
Archana
A
Mehta
Amee
A
Sahu
Mukesh
M
Awasthi
Sanjay
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000447:Aldehydes / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D002384:Catalysis; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006241:Haptens; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D008081:Liposomes; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes; D003432:Cross-Linking Reagents; D020690:GTPase-Activating Proteins; D006241:Haptens; D008081:Liposomes; C095637:RALBP1 protein, human; D011994:Recombinant Proteins; C030190:S-(2,4-dinitrophenyl)glutathione; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D005978:Glutathione; C027576:4-hydroxy-2-nonenal