Abstract
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.
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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Adenosine Triphosphatases / biosynthesis
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Adenosine Triphosphatases / isolation & purification
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Adenosine Triphosphatases / metabolism*
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Adenosine Triphosphate / pharmacology*
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Animals
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Azides / pharmacology
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Carrier Proteins / biosynthesis
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Carrier Proteins / isolation & purification
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Carrier Proteins / metabolism*
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Cell Line
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Cell Membrane / enzymology
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Cell Survival / drug effects
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Colchicine / toxicity
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Cricetinae
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Cricetulus
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Dactinomycin / toxicity
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Drug Interactions
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Drug Resistance
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Electrophoresis, Disc
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Kinetics
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Lung
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Membrane Glycoproteins / biosynthesis
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Membrane Glycoproteins / isolation & purification
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Membrane Glycoproteins / metabolism*
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Membrane Proteins / biosynthesis
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Membrane Proteins / isolation & purification
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Ouabain / pharmacology
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Sodium Azide
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Verapamil / pharmacology*
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Vincristine / toxicity
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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Azides
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Carrier Proteins
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Membrane Glycoproteins
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Membrane Proteins
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Dactinomycin
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Ouabain
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Vincristine
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Adenosine Triphosphate
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Sodium Azide
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Verapamil
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Adenosine Triphosphatases
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Colchicine