title: Identification of multiple binding sites for substrate transport in bovine organic anion transporting polypeptide 1a2.
abstract: Organic anion transporting polypeptides (OATP) have been extensively recognized as key determinants of absorption, distribution, metabolism, and excretion of various drugs because of their broad substrate specificity, wide tissue distribution, and the involvement of drug-drug interaction. As the first cloned human OATP, OATP1A2 has been found to transport a wide spectrum of endogenous and exogenous compounds. Bovine Oapt1a2 shared high homology with the human transporter and is considered as its functional ortholog. In the present study, we expressed bovine Oatp1a2 in human embryonic kidney 293 cells and found that, unlike human OATP1A2, the transport of estrone-3-sulfate (E-3-S) exhibited biphasic saturation kinetics. The K(m) values are 0.25 ± 0.08 and 46.6 ± 18.5 µM, and V(max) values were 24.5 ±4.4 and 375 ± 142 pmol/mg protein/min for high- and low-affinity sites, respectively, suggesting the presence of multiple binding sites. Further study on other Oatp1a2 substrates showed that the high affinity component for E-3-S is responsible for the interaction with taurocholate, bromsulphthalein, and rifampicin and is sensitive to proton concentration change, whereas the low affinity binding site is only involved in the binding of the antitumor drug methotrexate and had no response to change of pH.
authors.affiliation: College of Life Science and Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou, China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Liu
authors.forename: Jiujiu
authors.initials: J
authors.lastname: Huang
authors.forename: Yongxue
authors.initials: Y
authors.lastname: Sun
authors.forename: Kai
authors.initials: K
authors.lastname: Zhan
authors.forename: Zheren
authors.initials: Z
authors.lastname: Zhang
authors.forename: Mei
authors.initials: M
authors.lastname: Hong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D004970:Estrone / Q000031:analogs & derivatives* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism; D027361:Organic Anion Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012293:Rifampin / Q000378:metabolism; D013448:Sulfobromophthalein / Q000378:metabolism; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
chemical_list: D027361:Organic Anion Transporters; D011994:Recombinant Proteins; D013448:Sulfobromophthalein; D004970:Estrone; D013656:Taurocholic Acid; C017296:estrone sulfate; D012293:Rifampin; D008727:Methotrexate