Human PGP (MDR1) Membranes/ATPase Assay - FAQs

What are some of the applications of the PGP membranes?
The human PGP membrane product is a valuable pre-screening tool for determining if a drug interacts in any way with PGP. The ATPase assay provides a rapid, colorimetric, compound- independent measure of the concentration-dependence of any interaction of a drug with PGP. The membranes can potentially be used in uptake and ligand binding assays. However, methods for these applications are not currently available from BD Gentest.

Can the ATPase assay determine if a drug is a PGP substrate?
The ATPase assay can determine whether or not a drug stimulates PGP-ATPase activity and if the interaction demonstrates concentration-dependent saturation, which would suggest that the drug is a substrate for PGP transport. If a drug does not stimulate ATPase activity, the assay can still determine if the drug interacts with PGP, as an inhibitor of the ATPase activity stimulated by a known substrate, such as verapamil.

If a drug stimulates PGP-ATPase activity, will it be transported by PGP?
While ATP hydrolysis is required for drug transport , the ATPase assay does not directly measure drug transport (e.g. drugs can stimulate ATPase without being transported). PGP transport can be measured in vesicle uptake or monolayer permeability assays. BD Gentest has developed a stable mammalian cell system with cDNA-expressed human PGP. Our services with this system offer a direct measure of a drug’s transport across polarized cell monolayers. Coupled with the ATPase assay, the cell transport assay can further define the nature of a drug’s interaction with PGP.

How many ATPase assays can be performed with a vial of human PGP membranes?
Each vial contains enough material for 60 assay points using the conditions specified in the human PGP-ATPase assay method.

Does GENTEST sell control membranes for the human PGP membranes ?
Yes. We sell a membrane preparation (BD Gentest catalog no. 453200 (Old K200)) from insect cells infected with wild type virus, as a negative control for recombinant ABC transporters.

What is the suggested wavelength to use when performing the ATPase assay?
The absorption maximum of the phosphomolybdate chromophore produced in the reaction is at approximately 850nm. However, wavelengths between 630 and 850nm can be used. See Druekes et al., (1995) Analyt. Biochem. 230: 173)

Which organic solvents can be used with the membranes without affecting the assay results?
BD Gentest has tested acetonitrile, DMSO, ethanol, and methanol in the ATPase assay. Our data indicates that these solvents can be present up to 1% (in the final 60ul reaction) without significantly affecting the membrane activity.