Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1
Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1
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DOI：
10.1046/j.1432-1327.2000.01405.x
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发表时间：
2000-06-01
期刊：
影响因子：
--
通讯作者：
Bailey, PD
中科院分区：
文献类型：
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作者：
Meredith, D;Temple, CS;Bailey, PD
利用非洲爪蟾表达系统，研究了哺乳动物肠道肽转运蛋白PepT 1与一些氨基酸和肽衍生物的结合亲和力。比较了一系列封闭氨基酸，即N-乙酰基-Phe（Ac-Phe）、Phe-酰胺（Phe-NH 2）、N-乙酰基-Phe-酰胺（Ac-Phe-NH 2）和母体化合物Phe抑制肽[H-3]-D-Phe-L-Gln摄取的功效。在一组等效实验中，将封闭肽Ac-Phe-Tyr、Phe-Tyr-NH 2和Ac-Phe-Tyr-NH 2与母体化合物Phe-Tyr进行比较。比较氨基酸和衍生物，只有Ac-Phe是肽摄取的有效抑制剂（Ki = 1.81 +/- 0.37 mM）。Ac-Phe-NH 2与PepT 1的相互作用非常弱（Ki = 16.8 +/-5.64 mM）; Phe和Phe-NH 2均不以可测量的亲和力与PepT 1相互作用。对于二肽和衍生物，最高亲和力相互作用是与Phe-Tyr（Ki = 0.10 +/-0.04mM），这并不令人惊讶。封闭的C-末端肽Phe-Tyr-NH 2也以相对高的亲和力（Ki = 0.94 +/- 0.38 mM）与PepT 1相互作用。Ac-Phe-Tyr和Ac-Phe-Tyr-NH 2均与PepT 1弱相互作用（Ki分别为8.41 +/- 0.11和9.97 +/- 4.01 mM）。结果表明，N-末端是二肽和三肽的主要结合位点。用Ala-Ala-Ala的四种立体异构体进行的额外实验支持这一结论，并使我们提出组氨酸残基参与结合二肽的C-末端。此外，还提出了PepT 1的底物结合模型。
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 [H-3]-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 (K-i = 1.81 +/- 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 measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (K-i = 0.10 +/- 0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively 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 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.