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A new method to measure P-gp (ABCB1) activity.
Masatoshi Masuda1, Takaharu Mizutani
1Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedo-ri, Mizuho, Nagoya 467-8603, Japan.
Drug Metabolism Letters
|April 10, 2009
Summary
A new, sensitive method measures P-glycoprotein (P-gp) activity using radiolabeled ATP and charcoal adsorption. This technique simplifies P-gp assays and reduces membrane requirements for drug transport studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- P-glycoprotein (P-gp) is a key efflux transporter involved in drug resistance.
- Accurate measurement of P-gp activity is crucial for drug development and understanding drug disposition.
- Existing methods for assessing P-gp activity can be resource-intensive.
Purpose of the Study:
- To develop a simplified and sensitive assay for measuring P-glycoprotein (P-gp) activity.
- To provide a cost-effective alternative to conventional P-gp activity assays.
- To establish a method applicable to other ATP-binding cassette (ABC) transporters.
Main Methods:
- Utilized [gamma-(32)P]ATP as a substrate and charcoal for adsorption of inorganic phosphate.
- Incubated P-gp membranes with [gamma-(32)P]ATP and drug, followed by charcoal treatment.
- Measured radioactivity in the supernatant containing inorganic (32)P using a radio-image analyzer.
Main Results:
- The developed method demonstrated high sensitivity for measuring P-gp activity.
- This assay requires significantly less P-gp membrane material compared to traditional methods.
- The method proved effective in phosphate buffer and applicable to other ABC transporters.
Conclusions:
- A novel, easy, and sensitive assay for P-gp activity has been established.
- This method offers advantages in terms of reduced sample requirements and potential broader applicability.
- The assay provides a valuable tool for research involving P-gp and other ABC transporters.
