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Development and characterization of the organic anion transporter column for on line studies of drug‐transporter affinities
Author(s) -
Kimura Tomoko,
Moaddel Ruin,
Wainer Irving W.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1188-d
Subject(s) - organic anion transporter 1 , chemistry , chromatography , high performance liquid chromatography , affinity chromatography , transporter , membrane , in vitro , in vivo , ligand (biochemistry) , affinities , ion chromatography , biochemistry , receptor , enzyme , biology , microbiology and biotechnology , gene
Purpose: To develop liquid chromatographic columns containing immobilized organic anion transporters (hOAT1, hOAT2) Method: Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane liquid chromatographic stationary phase. The resulting stationary phases were packed into a HR 5/2 glass columns to yield a 150 cm x 5 mm (ID) chromatographic beds and placed in a HPLC system containing an on‐line scintillation detector. Frontal affinity chromatography studies were performed using the marker ligand [ 3 H]‐adefovir (OAT1) or [ 14 C]‐ p ‐aminohippurate (OAT2) and increasing concentrations known ligands as the displacers. Results: The binding affinities (Kd) for five OAT1 ligands and two OAT2 ligands were determined by frontal displacement chromatography on the respective columns. The Kd values obtained for the OAT1 ligands correlated with literature values (r 2 = 0.776, p = 0.0479). The Kds for the OAT2 ligands were also comparable to reported values; e.g, the chromatographically determined Kd for AZT was 81microM versus 26.8microM in literature. Conclusions: The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.