A rapid and inexpensive method to assay transport of short chain peptides across intestinal brush-border membrane vesicles from the European eel (Anguilla anguilla) |
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| Authors: | T VERRI A DANIELI S BAKKE A ROMANO A BARCA I RØNNESTAD M MAFFIA & C STORELLI |
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| Institution: | Department of Biological and Environmental Sciences and Technologies, University of Salento (formerly University of Lecce), Lecce, Italy;; Department of Biology, University of Bergen, Bergen, Norway |
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| Abstract: | Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla ) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3'-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na+ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na+-independent, H+-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( K m,app ～ 1.5 mmol L?1) and that within the 0.1–10 mmol L?1 peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H+/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay. |
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| Keywords: | cyanine dye di/tripeptide transport DiS-C2(5) membrane potential PepT1 Slc15A1 |
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