title: ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.
abstract: 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.
authors.affiliation: SOLVO Biotechnology Inc. Central Hungarian Innovations Center, Gyár u. 2, H-2040 Budaörs, Hungary.
authors.forename: Hristos
authors.initials: H
authors.lastname: Glavinas
authors.forename: Emese
authors.initials: E
authors.lastname: Kis
authors.forename: Akos
authors.initials: A
authors.lastname: Pál
authors.forename: Rita
authors.initials: R
authors.lastname: Kovács
authors.forename: Márton
authors.initials: M
authors.lastname: Jani
authors.forename: Erika
authors.initials: E
authors.lastname: Vági
authors.forename: Eva
authors.initials: E
authors.lastname: Molnár
authors.forename: Száva
authors.initials: S
authors.lastname: Bánsághi
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Kele
authors.forename: Tamás
authors.initials: T
authors.lastname: Janáky
authors.forename: György
authors.initials: G
authors.lastname: Báthori
authors.forename: Oliver
authors.initials: O
authors.lastname: von Richter
authors.forename: Gerrit-Jan
authors.initials: GJ
authors.lastname: Koomen
authors.forename: Péter
authors.initials: P
authors.lastname: Krajcsi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010053:Ovary / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D017510:Protein Folding; D018411:Spodoptera; D014638:Vanadates / Q000494:pharmacology
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D004364:Pharmaceutical Preparations; D004970:Estrone; D014638:Vanadates; D004958:Estradiol; D000251:Adenosine Triphosphatases; C070990:vanadate-sensitive ATPase; C017296:estrone sulfate