Pgp-Glo A Luminescent Method for Measuring P-Glycoprotein ATPase Activity Scientific...
Pgp-Glo A Luminescent Method for Measuring P-Glycoprotein ATPase Activity Scientific Poster
James J. Cali and Dongping Ma, Promega Corporation, Madison, WI, USA
We have developed a luminescent method for detecting drug-dependent changes in the ATPase activity of the multi-drug transporter P-glycoprotein (Pgp, MDR1 or ABCB1). Pgp is an ATP-dependent efflux pump for a wide range of drugs that plays an important role in multi-drug resistance and certain adverse drug-drug interactions. Drugs that are transported by Pgp can be identified as stimulators of its ATPase activity. Our luminescent method relies on the ATP dependence of the light-generating reaction of firefly luciferase. After a pool of ATP is first exposed to the Pgp ATPase ATP consumption is detected as a decrease in luminescence from a second reaction with firefly luciferase. The quantity of ATP consumed can be interpolated from a luciferase/ATP standard curve and from this the rate of ATPase activity calculated. We used a stabilized mutant of the luciferase enzyme in a formulation that provided stable, glow-type signals from multiwell plates. The method detected the dose-dependent stimulation of recombinant human Pgp ATPase activity by verapamil and inhibition of this activity by cyclosporin-A. This simple add and read format is well suited for screening applications.
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