Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2

Abstract

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1, hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [ 3 H]-adefovir for the hOAT1 and [ 14 C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2= 0.688 (p < 0.05) and r2=0.9967 (p<0.0001), respectively. 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.

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

“…The dissociation constants, K d ’s, for the displacer ligands were determined using a previously reported approach [28]. The experimental paradigm is based upon the effect of escalating approach of a competitive binding ligand on the retention volume.…”

Synthesis and characterization of a SIRT6 open tubular column: Predicting deacetylation activity using frontal chromatography

“…The dissociation constants, K d , for the displacer ligands were determined using a previously reported approach [14]. The experimental paradigm is based upon the effect of escalating approach of a competitive binding ligand on the retention volume.…”

Organic Anion Transporters and Their Implications in Pharmacotherapy

“…hOAT2 displayed very high affinity for transport of 5-fluorouracil, MTX, and paclitaxel (Taxol) (Kobayashi et al, 2005). Some conflicting data exist for rOAT2 with relation to MTX transport (Sekine et al, 1998;Sun et al, 2001;Kimura et al, 2007).…”