title: Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.
abstract: The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.
authors.affiliation: Department of Human Anatomy & Genetics, South Parks Road, Oxford, UK. david.meredith@bristol.ac.uk
authors.forename: D
authors.initials: D
authors.lastname: Meredith
authors.forename: C S
authors.initials: CS
authors.lastname: Temple
authors.forename: N
authors.initials: N
authors.lastname: Guha
authors.forename: C J
authors.initials: CJ
authors.lastname: Sword
authors.forename: C A
authors.initials: CA
authors.lastname: Boyd
authors.forename: I D
authors.initials: ID
authors.lastname: Collier
authors.forename: K M
authors.initials: KM
authors.lastname: Morgan
authors.forename: P D
authors.initials: PD
authors.lastname: Bailey
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D005260:Female; D009865:Oocytes / Q000378:metabolism; D000074011:Peptide Transporter 1; D010455:Peptides / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D027981:Symporters; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; D010455:Peptides; D027981:Symporters; D010649:Phenylalanine; C020457:phenylalanine amide; C044228:N-acetylphenylalanine