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Abstract for Pgp-Glo™: A Luminescent Method for Measuring
P-Glycoprotein ATPase Activity
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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