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A New Method to Measure P-gp (ABCB1) Activity

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We have developed an easy and sensitive method to measure P-glycoprotein (P-gp) activity using [γ-32P]ATP and charcoal. This method utilizes the nature of adsorption of organic phosphate to charcoal. The standard assay method is as follows: the reaction mixture (20 μl) of 1 mM [γ-32P]ATP (1 Ci/mol), 2.5 μg P-gp membranes, and the drug was incubated for 30 min, and 50 μl of 10% charcoal suspension in 0.1 M phosphate buffer at pH7.3 was then added to the mixture. The solution was centrifuged and the supernatant (20 μl) in duplicate containing inorganic 32P was spotted onto filter paper, and radioactivity was measured by radio-image analyzer. This method can reduce the amount of P-gp membrane compared to the conventional method utilizing coloring of the inorganic phosphate-molybdate reaction. This method is also applicable to other ATP-binding cassette (ABC) transporters in phosphate buffer.





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Keywords: 32P; ABCB1; ATP; MDR1; P-glycoprotein; charcoal

Document Type: Research Article

Publication date: December 1, 2007

More about this publication?
  • Drug Metabolism Letters publishes short papers on major advances in all areas of drug metabolism and disposition. The emphasis will be on publishing quality papers very rapidly. Letters will be processed rapidly by taking full advantage of the Internet technology for both the submission and review of manuscripts. The journal covers the following areas:

    In vitro systems including CYP-450; enzyme induction and inhibition; drug-drug interactions and enzyme kinetics; pharmacokinetics, toxicokinetics, species scaling and extrapolations; P-glycoprotein and transport carriers; target organ toxicity and interindividual variability; drug metabolism and disposition studies; extrahepatic metabolism; phase I and phase II metabolism; recent developments for the identification of drug metabolites, reactive intermediate and glutathione conjugates.
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