Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2.
This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and 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 [3H]-adefovir for the hOAT1 and [14C]-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.
Kimura
T
T
Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
Perry
J
J
Anzai
N
N
Pritchard
J B
JB
Moaddel
R
R
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D027362:Organic Anion Transport Protein 1 / Q000737:chemistry*; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry*; D011485:Protein Binding
D027362:Organic Anion Transport Protein 1; D029362:Organic Anion Transporters, Sodium-Independent; C490280:SLC22A7 protein, human