RT Journal Article
SR Electronic
T1 ABCG2 (Breast Cancer Resistance Protein/Mitoxantrone Resistance-Associated Protein) ATPase Assay: A Useful Tool to Detect Drug-Transporter Interactions
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1533
OP 1542
DO 10.1124/dmd.106.014605
VO 35
IS 9
A1 Glavinas, Hristos
A1 Kis, Emese
A1 Pál, Ákos
A1 Kovács, Rita
A1 Jani, Márton
A1 Vági, Erika
A1 Molnár, Éva
A1 Bánsághi, Száva
A1 Kele, Zoltán
A1 Janáky, Tamás
A1 Báthori, György
A1 von Richter, Oliver
A1 Koomen, Gerrit-Jan
A1 Krajcsi, Péter
YR 2007
UL http://dmd.aspetjournals.org/content/35/9/1533.abstract
AB The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance. The American Society for Pharmacology and Experimental Therapeutics