Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1
Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1
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DOI：
10.1074/jbc.273.7.3861
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发表时间：
1998-02-13
影响因子：
4.8
通讯作者：
Neubert, K
中科院分区：
文献类型：
--
作者：
Brandsch, M;Thunecke, F;Neubert, K
本研究旨在确定肠道H+/肽同调体PEPT1是否区分底物的肽键构象，我们合成了一种修饰的二肽，其中肽键被等构硫氧肽键取代，该Ala-Pro衍生物Ala-psi[CS-N]-Pro在水溶液中以顺式和反式构象的混合物存在，具有低的顺式/反式异构化率，该化合物被PEPT1以高亲和力识别。在含有96%反式构象的溶液中，Ala-psi[CS-N]- pro对cco -2细胞中放射性标记甘氨酸肌氨酸摄取的抑制K-i值为0.30 +/- 0.02 mM，而在含有62%反式构象的摄取介质中，其Ri值为0.51 +/- 0.02 mM。我们得出结论，只有反式构象与转运系统相互作用。在第二种方法中，通过分析运输后Caco-2细胞的细胞内含量以及使用毛细管电泳分析细胞外培养基的组成，研究了Ala-psi[CS-N]-Pro的构象特异性摄取。从摄取介质中62%的反式构象比例增加到细胞内的92%，这是H+/肽共转运系统选择性结合和转运肽衍生物的反式构象的第一个直接证据。
This study was initiated to determine whether the intestinal H+/peptide symporter PEPT1 differentiates between the peptide bond conformers of substrates, We synthesized a modified dipeptide where the peptide bond is replaced by the isosteric thioxo peptide bond, The Ala-Pro derivative Ala-psi[CS-N]-Pro exists as a mixture of cis and trans conformation in aqueous solution and is characterized by a low cis/trans isomerization rate, The compound was recognized by PEPT1 with high affinity, The K-i value of Ala-psi[CS-N]-Pro for the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2 cells was 0.30 +/- 0.02 mM, determined in solution with 96% trans conformation, In contrast, the Ri value was 0.51 +/- 0.02 mM when uptake media with 62% trans conformer were used, We conclude that only the trans conformer interacts with the transport system. From our data, a significant affinity of the cis conformer at PEPT1 cannot be derived, In a second approach, conformer-specific uptake of Ala-psi[CS-N]-Pro was studied by analyzing the intracellular content of Caco-2 cells following transport as well as the composition of the extracellular medium using capillary electrophoresis. The percentage of trans conformer that was 62% in the uptake medium increased to 92% inside the cells, This is the first direct evidence that an H+/peptide cotransport system selectively binds and transports the trans conformer of a peptide derivative.