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Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1

Meredith, David, Temple, Catherine S., Guha, Nishan, Sword, Corinna J., Boyd, C. A. Richard, Collier, Ian D., Morgan, Keith M., Bailey, Patrick D.
European journal of biochemistry 2000 v.267 no.12 pp. 3723-3728
Xenopus laevis, binding capacity, binding sites, dipeptides, histidine, mammals, models, peptide transporters, stereoisomers, tripeptides
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.