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The Journal of Experimental Biology 206, 705-714 (2003)
doi: 10.1242/jeb.00145

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

M. Maffia^1,*, A. Rizzello¹, R. Acierno¹, T. Verri¹, M. Rollo¹, A. Danieli¹, F. Döring², H. Daniel² and C. Storelli¹

¹ Laboratory of General Physiology, Department of Biological and Environmental Science and Technology, University of Lecce, strada prov. le Lecce-Monteroni, I-73100 Lecce, Italy
² Institute of Nutritional Sciences, Physiology and Biochemistry of Nutrition, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany

^* Author for correspondence (e-mail: m.maffia{at}physiology.unile.it)

Accepted 18 November 2002

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,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 D-Phe-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 0°C for the former and 25°C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Key words: brush-border membrane vesicle, Xenopus laevis oocyte, PepT1, H⁺/peptide cotransport, fish, intestine, Antarctic fish