H+/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H+/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K-m,K-app) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H+/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated DPhe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H+/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0degreesC for the former and 25degreesC for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus

MAFFIA, Michele;RIZZELLO, Antonia;ACIERNO, Raffaele;VERRI, Tiziano;DANIELI, Antonio;STORELLI, Carlo
2003

Abstract

H+/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H+/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K-m,K-app) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H+/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated DPhe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H+/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0degreesC for the former and 25degreesC for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11587/300881
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