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 micrography confirmed the formation of unilamellar vesicles with an average radius of 0.25 micron. Intravesicular volume estimated by incorporation of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 microL/mL reconstitution solution. Accumulation of the glutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposomes was increased in the presence of ATP as compared to equimolar ADP or 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 to the osmolarity of the assay medium, and temperature dependent. The energy 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 (Vmax of 433 +/- 20 nmol/min/mg of protein, KmATP = 2.4 +/- 0. 3 mM and KmDNP-SG = 36 +/- 5 microM) as well as DOX (Vmax = 194 +/- 19 nmol/min/mg of protein, KmATP = 2.5 +/- 0.6 mM and KmDOX = 2.4 +/- 0.7 microM). 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 inhibitor of DNP-SG transport with Kis of 1.2 +/- 0.2 microM and DNP-SG was found to be a competitive inhibitor of DOX transport with Kis of 13.3 +/- 2.6 microM.
Awasthi
S
S
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas, USA. sawasthi@utmb.edu
Singhal
S S
SS
Pikula
S
S
Piper
J T
JT
Srivastava
S K
SK
Torman
R T
RT
Bandorowicz-Pikula
J
J
Lin
J T
JT
Singh
S V
SV
Zimniak
P
P
Awasthi
Y C
YC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D004317:Doxorubicin / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D009997:Osmotic Pressure; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids / Q000378:metabolism / Q000648:ultrastructure; D013696:Temperature
D002352:Carrier Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C086860:glutathione transporter; C020485:proteoliposomes; C030190:S-(2,4-dinitrophenyl)glutathione; C024990:asolectin; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C068387:S-(dinitrophenyl)glutathione ATPase; D005978:Glutathione; D019803:Glutathione Disulfide