Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.
We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte 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. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.
Singhal
S S
SS
Department of Chemistry and Biochemistry, The University of Texas at Arlington, 76019-0065, USA.
Singhal
J
J
Cheng
J
J
Pikuła
S
S
Sharma
R
R
Zimniak
P
P
Awasthi
Y C
YC
Awasthi
S
S
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000328:Adult; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism*; D004317:Doxorubicin / Q000493:pharmacokinetics; D004910:Erythrocyte Membrane / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008297:Male; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D020725:ral GTP-Binding Proteins / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D008081:Liposomes; C095637:RALBP1 protein, human; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D004317:Doxorubicin; D000251:Adenosine Triphosphatases; D020725:ral GTP-Binding Proteins