Abe H, Satoh M, Miyauchi S, Shuto S, Matsuda A, Kamo N
Laboratory of Biophysical Chemistry and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812 Japan.
Bioconjug Chem. 1999 Jan-Feb;10(1):24-31. doi: 10.1021/bc980049i.
Dipeptide transporters in small intestine have a very wide substrate specificity, so that the transporter sometimes serves as a carrier for peptide-like compounds. We have synthesized dipeptide analogues conjugated at an epsilon-amino group of Lys in Val-Lys or Lys-Sar with fluorescent compounds such as fluorescein isothiocyanate and coumarin-3-carboxylic acid. Uptakes of these peptide analogues were examined by measuring intracellular accumulations into monolayers of the human intestinal epithelial cell line Caco-2 expressing the dipeptide transporter PEPT1. Kinetic analysis and effects of addition either of uncoupler (protonophore) or by Gly-Sar, one of the good substrates of PEPT1, revealed that fluorescent dipeptides were taken up by passive diffusion. In contrast, these analogues remarkably inhibited the Gly-Sar uptake by Caco-2 cells. Among the fluorescent analogues synthesized in this paper, Val-Lys(Flu) was the most potent competitive inhibitor against the Gly-Sar uptake with an inhibition constant of 5 microM. This value is the smallest among those ever reported: Val-Lys(Flu) has the highest affinity for PEPT1 among chemicals ever reported. The importance of the hydrophobic part of the substrate was pointed out.
小肠中的二肽转运体具有非常广泛的底物特异性，因此该转运体有时可作为肽类化合物的载体。我们已合成了在缬氨酸 - 赖氨酸（Val - Lys）或赖氨酸 - 肌氨酸（Lys - Sar）中赖氨酸的ε - 氨基处与荧光化合物（如异硫氰酸荧光素和香豆素 - 3 - 羧酸）共轭的二肽类似物。通过测量这些肽类似物在表达二肽转运体PEPT1的人肠上皮细胞系Caco - 2单层细胞中的细胞内积累来检测其摄取情况。动力学分析以及添加解偶联剂（质子载体）或PEPT1的良好底物之一甘氨酰 - 肌氨酸（Gly - Sar）的影响表明，荧光二肽是通过被动扩散被摄取的。相反，这些类似物显著抑制Caco - 2细胞对甘氨酰 - 肌氨酸的摄取。在本文合成的荧光类似物中，缬氨酸 - 赖氨酸（荧光素）（Val - Lys(Flu)）是对甘氨酰 - 肌氨酸摄取最有效的竞争性抑制剂，抑制常数为5 microM。该值是迄今报道的最小的值：在迄今报道的化学物质中，缬氨酸 - 赖氨酸（荧光素）对PEPT1具有最高的亲和力。底物疏水部分的重要性也被指出。
