ATP-DEPENDENT HUMAN ERYTHROCYTE GLUTATHIONE-CONJUGATE TRANSPORTER - II - FUNCTIONAL RECONSTITUTION OF TRANSPORT ACTIVITY

Purified dinitrophenyl S-glutathione (DNP-SG) ATPase was reconstituted into artificial liposomes prepared from soybean asolectin. Electron m icrography confirmed the formation of unilamellar vesicles with an ave rage radius of 0.25 mu m. Intravesicular volume estimated by incorpora tion of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 mu L/mL reconstitution solution. Accumulation of the glutathione-c onjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposo mes was increased in the presence of ATP as compared to equimolar ADP of adenosine 5'-[beta,gamma-methylene] triphosphate tetralithium. ATP- dependent transmembrane movement of DOX and DNP-SG into DNP-SG ATPase- reconstituted vesicles was saturable with respect to time, sensitive t o the osmolarity of the assay medium, and temperature dependent. The e nergy of activation was found to be 12 and 15 kcal/mol for DNP-SG and DOX, respectively. Optimal temperature for transport was 37 degrees C. Saturable transport was demonstrated for DNP-SG (V-max of 433 +/- 20 nmol/min/mg of protein, K-mATP = 2.4 +/- 0.3 mM and KmDNP-SG = 36 +/- 5 mu M) as well as DOX (V-max = 194 +/- 19 nmol/min/mg of protein, K-m ATP = 2.5 +/- 0.6 mM and K-mDOX = 2.4 +/- 0.7 mu M). The kinetic data for both DNP-SG and DOX transport were consistent with a random bi-bi sequential reaction mechanism. DOX was found to be a competitive inhib itor of DNP-SG transport with K-is of 1.2 +/- 0.2 mu M and DNP-SG was found to be a competitive inhibitor of DOX transport with K-is of 13.3 +/- 2.6 mu M.