We have recently shown that RLIP76, a ral-bind ing GTPase ac ti vat ing pro tein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi eta/., Biochemistry39l9327l2000).TransportfunctionofRLIP76 was found to be in tact de spite con sid er able proteolytic frag men ta tion in prep a ra tions used for those stud ies, sug gest ing ei ther that the re sid ual in tact RLIP76 was re spon si ble for trans port ac tiv ity, or that the trans port ac tiv ity could be re con sti tuted by fragments of RLIP76. If the for mer were true, in tact RLIP76 would have a much higher spe cific ac tiv ity for ATP-hydrolysis than the frag mented pro tein. We have ad dressed this ques tion by com par ing trans port prop er ties of re com bi nant RLIP76 and hu man eryth ro cyte mem brane RLIP76 pu ri fied in buff ers treated with either 100 or 500uM serine pro te ase in hib i tor, PMSF. The pu rity and iden tity of re com bi nant and hu man eryth ro cyte RLIP76 was es tab lished by SDS/PAGE and West ern-blot anal y sis. These studies con firmed the or i gin of the 38 kDa pro tein, pre vi ously re ferred to as DNP-SG ATPase, from RLIP76. Higher PMSF con cen tra tion re sulted in lower yield of the 38 kDa band and higher yield of in tact RLIP76 from both hu man and re com bi nant source. In con trast, the sub strate-stimulated ATPase ac tiv ity in pres ence of DNP-SG, doxorubicin, daunorubicin, or colchicine were un af fected by in creased PMSF; similarly, ATP-dependent trans port of doxorubicin in proteo liposomes re con sti tuted with RLIP76 was un af fected by higher PMSF. These re sults in di cated that lim ited proteolysis by serine pro teas es does not ab ro gate the trans port func tion of RLIP76. Com parison of trans port ki net ics for daunorubicin be tween re com bi nant vs hu man erythrocyte RLIP76 re vealed higher spe cific ac tiv ity of trans port for tis sue pu ri fied RLIP76, in di cat ing that ad di tional fac tors pres ent in tis sue pu ri fied RLIP76 can mod u late its transport activity.
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