Abstract
P-glycoprotein was purified from multidrug-resistant Chinese hamster ovary CHRB30 cells by a combination of anion exchange and immunoaffinity chromatography. The P-glycoprotein was about 90% pure and had a Vmax for ATP hydrolysis in detergent solution of 321 nmol/min/mg with a Km of 0.94 mM. The ATPase activity was inhibited by low concentrations of vanadate and N-ethylmaleimide, but unaffected by azide or ouabain. When the purified P-glycoprotein was reconstituted into phospholipid bilayer membranes, the ATPase activity became highly stimulated by several chemosensitizers and drugs involved with multidrug resistance. Verapamil, a potent chemosensitizer, increased the Vmax for ATP hydrolysis by 22-fold and the Km for ATP by 5.4-fold. This effect of verapamil on P-glycoprotein has not previously been observed. These results demonstrate that purified P-glycoprotein has an intrinsic ATPase activity with unique properties. This activity appears sufficient to account for the ATP-dependent reduction in intracellular drug accumulation of P-glycoprotein-expressing multidrug-resistant cells.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
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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Amino Acid Sequence
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Animals
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Antibodies, Monoclonal
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Azides / pharmacology
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CHO Cells
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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 Membrane / drug effects
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Cell Membrane / metabolism
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Chromatography, Affinity
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Chromatography, DEAE-Cellulose
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Clone Cells
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Cricetinae
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Drug Resistance*
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Electrophoresis, Polyacrylamide Gel
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Ethylmaleimide / pharmacology
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Kinetics
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Liposomes
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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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Molecular Sequence Data
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Ouabain / pharmacology
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Proteolipids / metabolism
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Ultrafiltration
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Vanadates / pharmacology
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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Antibodies, Monoclonal
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Azides
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Carrier Proteins
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Liposomes
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Membrane Glycoproteins
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Proteolipids
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proteoliposomes
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Vanadates
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Ouabain
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Adenosine Triphosphatases
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Ethylmaleimide