A Novel Inhibitor of the Mammalian Peptide Transporter PEPT1
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- 作者：Ilka Knü ; tter ; Stephan Theis ; Bianka Hartrodt ; Ilona Born ; Matthias Brandsch ; Hannelore Daniel ; and Klaus Neubert
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：April 10, 2001
- 年：2001
- 卷：40
- 期：14
- 页码：4454 - 4458
- 全文大小：63K
- 年卷期：v.40,no.14(April 10, 2001)
- ISSN：1520-4995
文摘
This study was initiated to develop inhibitors of the intestinal H+/peptide symporter. We provideevidence that the dipeptide derivative Lys[Z(NO2)]-Pro is an effective competitive inhibitor of mammalianPEPT1 with an apparent binding affinity of 5-10 
M. Characterization of the interaction of Lys[Z(NO2)]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of humanCaco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopuslaevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLCanalysis of Lys[Z(NO2)]-Pro in cells, and electrophysiological techniques, we unequivocally show thatLys[Z(NO2)]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides foruptake into cells, but is not transported itself. Lack of transport was substantiated by the absence ofLys[Z(NO2)]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of anypositive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO2)]-Pro canbind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expressionsystem by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findingsserve as a starting point for the identification of the substrate binding domain in the PEPT1 protein aswell as for studies on the physiological and pharmacological role of PEPT1.
M. Characterization of the interaction of Lys[Z(NO2)]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of humanCaco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopuslaevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLCanalysis of Lys[Z(NO2)]-Pro in cells, and electrophysiological techniques, we unequivocally show thatLys[Z(NO2)]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides foruptake into cells, but is not transported itself. Lack of transport was substantiated by the absence ofLys[Z(NO2)]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of anypositive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO2)]-Pro canbind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expressionsystem by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findingsserve as a starting point for the identification of the substrate binding domain in the PEPT1 protein aswell as for studies on the physiological and pharmacological role of PEPT1.