Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal.
Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with K(m) values of 1.4mM and 2.5 microM, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.
Sharma
Rajendra
R
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0647, USA.
Sharma
Abha
A
Yang
Yusong
Y
Awasthi
Sanjay
S
Singhal
Sharad S
SS
Zimniak
Piotr
P
Awasthi
Yogesh C
YC
D018528:ATP-Binding Cassette Transporters; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005374:Filtration / Q000379:methods; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D011510:Proteolipids / Q000378:metabolism*; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D011510:Proteolipids; C095637:RALBP1 protein, human; C020485:proteoliposomes; D014316:Tritium; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D005978:Glutathione; C027576:4-hydroxy-2-nonenal