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 dox orubicin (DOX) (S. Awasthi et al., Biochemistry 39, 9327, 2000). Transport function of RLIP78 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either tha t the residual intact RLIP76 was responsible for transport activity, or tha t the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activ ity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLTP76 purified in buffers treated with either 100 or 500 muM serine protease inhibitor, PMSF. The purity and identity of recombi nant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-b lot analysis. These studies confirmed the origin of the 38 kDa protein, pre viously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentratio n resulted in lower yield of the 38 kDa band and higher yield of intact RLI P76 from both human and recombinant source. In contrast, the substrate-stim ulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent tra nsport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaf fected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Compar ison of transport kinetics for daunorubicin between recombinant vs human er ythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purif ied RLIP76 can modulate its transport activity.