title: Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.
abstract: In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.
authors.affiliation: Biozentrum of the Martin-Luther-University Halle-Wittenberg, Halle, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Knütter
authors.forename: Bianka
authors.initials: B
authors.lastname: Hartrodt
authors.forename: Géza
authors.initials: G
authors.lastname: Tóth
authors.forename: Attila
authors.initials: A
authors.lastname: Keresztes
authors.forename: Gabor
authors.initials: G
authors.lastname: Kottra
authors.forename: Carmen
authors.initials: C
authors.lastname: Mrestani-Klaus
authors.forename: Ilona
authors.initials: I
authors.lastname: Born
authors.forename: Hannelore
authors.initials: H
authors.lastname: Daniel
authors.forename: Klaus
authors.initials: K
authors.lastname: Neubert
authors.forename: Matthias
authors.initials: M
authors.lastname: Brandsch
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism*; D011868:Radioisotopes; D051381:Rats; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D010455:Peptides; D011868:Radioisotopes; D006859:Hydrogen