Meredith D, Temple C S, Guha N, Sword C J, Boyd C A, Collier I D, Morgan K M, Bailey P D
Department of Human Anatomy & Genetics, South Parks Road, Oxford, UK.
Eur J Biochem. 2000 Jun;267(12):3723-8. doi: 10.1046/j.1432-1327.2000.01405.x.
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.
利用非洲爪蟾表达系统，研究了多种氨基酸和肽衍生物与哺乳动物肠道肽转运体PepT1的结合亲和力。比较了一系列封闭氨基酸，即N - 乙酰苯丙氨酸（Ac - Phe）、苯丙氨酰胺（Phe - NH2）、N - 乙酰苯丙氨酰胺（Ac - Phe - NH2）及其母体化合物苯丙氨酸（Phe）对肽[3H] - D - 苯丙氨酸 - L - 谷氨酰胺摄取的抑制效果。在一组等效实验中，将封闭肽Ac - Phe - Tyr、Phe - Tyr - NH2和Ac - Phe - Tyr - NH2与母体化合物Phe - Tyr进行了比较。比较氨基酸及其衍生物时，只有Ac - Phe是肽摄取的有效抑制剂（Ki = 1.81±0.37 mM）。Ac - Phe - NH2与PepT1的相互作用非常弱（Ki = 16.8±5.64 mM）；Phe和Phe - NH2与PepT1均未表现出可测量的亲和力相互作用。对于二肽及其衍生物，不出所料，最高亲和力相互作用发生在Phe - Tyr（Ki = 0.10±0.04 mM）。封闭C端的肽Phe - Tyr - NH2与PepT1也具有相对较高的亲和力相互作用（Ki = 0.94±0.38 mM）。Ac - Phe - Tyr和Ac - Phe - Tyr - NH2与PepT1的相互作用都很弱（分别为Ki = 8.41±0.11 mM和9.97±4.01 mM）。结果表明，N端是二肽和三肽的主要结合位点。对丙氨酸 - 丙氨酸 - 丙氨酸的四种立体异构体进行的额外实验支持了这一结论，并使我们提出组氨酸残基参与二肽C端的结合。此外，还提出了PepT1的底物结合模型。
