Modified amino acids and peptides as substrates for the intestinal peptidetransporter PepT1

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, usin g the Xenopus laevis expression system. A series of blocked amino acids, na mely 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 [H-3]-D-Phe-L-Gln. In an equivalent set of experi ments, 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 deriv atives, only Ac-Phe was an effective inhibitor of peptide uptake (K-i = 1.8 1 +/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (K-i = 16 .8 +/- 5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measura ble affinity. With the dipeptide and derivatives, unsurprisingly the highes t affinity interaction was with Phe-Tyr (K-i = 0.10 +/- 0.04 mM). The block ed C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relativ ely high affinity (K-i = 0.94 +/- 0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr- NH2 interacted weakly with PepT1 (K-i = 8.41 +/- 0.11 and 9.97 +/- 4.01 mM, respectively). The results suggest that the N-terminus is the primary bind ing site for both dipeptides and tripeptides. Additional experiments with f our stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to pr opose that a histidine residue is involved in binding the C-terminus of dip eptides. In addition, a substrate binding model for PepT1 is proposed.