Absence of cooperativity for MgATP and verapamil effects on the ATPase activity of P-glycoprotein containing membrane vesicles.
Purified membrane vesicles were prepared from Chinese Hamster lung fibroblasts expressing high amounts of P-glycoprotein (P-gp), which is responsible for the multidrug resistance. P-gp ATPase activity, characterized in the presence or absence of verapamil, had a Michaelian behavior for its MgATP dependence. Thus only one MgATP molecule should be sufficient for the catalytic cycle. With increasing verapamil concentrations, a bell-shape curve was observed for ATPase activity, with half-activation and -inhibition concentrations of 1.2 microM and 490 microM, respectively. No cooperativity for verapamil was detected. These results strongly suggest that P-gp functions as an active transporter, with a coupling stoichiometry of one MgATP molecule hydrolysed for one verapamil molecule transported.
Garrigos
M
M
Section de Biophysique des Protéines et des Membranes DBCM/CEA and URA 1290 CNRS, Gif/Yvette, France.
Belehradek
J
J
Mir
L M
LM
Orlowski
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D003078:Colchicine / Q000633:toxicity; D006224:Cricetinae; D003412:Cricetulus; D003609:Dactinomycin / Q000633:toxicity; D004347:Drug Interactions; D004351:Drug Resistance; D004589:Electrophoresis, Disc; D007700:Kinetics; D008168:Lung; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D010042:Ouabain / Q000494:pharmacology; D019810:Sodium Azide; D014700:Verapamil / Q000494:pharmacology*; D014750:Vincristine / Q000633:toxicity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D003609:Dactinomycin; D010042:Ouabain; D014750:Vincristine; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003078:Colchicine