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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

View larger version (51K): [in a new window]   Fig. 1. (A) Expression of PepT1-related mRNA in icefish and eel intestine by RT-PCR. The PepT1 isoform was detected in the intestine of icefish (lane 2) and eel (lane 4; positive control). The identity of each isoform was confirmed by PCR product size (570 bp) and sequencing (data not shown). Lanes 1 and 6: markers; lanes 3 and 5: RT-PCR control (RNA without reverse transcriptase). (B) Comparison of primary amino acid sequences of icefish and human PepT1 H+/peptide cotransporters. Asterisks indicate indentical amino acids; colons, conserved substitution (small, acidic, basal or hydroxyl+Amine+Basic, etc.); stops, semiconserved substitution.

View larger version (20K): [in a new window]   Fig. 2. (A) Peptide-dependent Acridine Orange fluorescence quenching in icefish intestine brush-border membrane vesicles (BBMVs). In addition to valinomycin, the cuvettes contained 100 mmol l-1 KCl (trace a); 100 mmol l-1 KCl + 20 mmol l-1 Gly-L-Pro (trace b); 100 mmol l-1 choline chloride (trace c); 100 mmol l-1 choline chloride + 20 mmol l-1 Gly-L-Pro (trace d). (B) Effects of exogenous dipeptides on H+ influx rate (trace d — trace c, N=5). (C) Dependence of the initial rate of H+ influx on increasing extravesicular Gly-L-Pro concentrations. Inset, the Woolf—Augustinsson—Hofstee plot of the experimental data (N=3). (D) Diethylpyrocarbonate (DEP) inhibition of proton accumulation in icefish intestinal BBMVs (N=4). Values were significant at *P<0.05; **P<0.01.

View larger version (26K): [in a new window]   Fig. 3. (A) [3H]-D-Phe-L-Ala uptake in oocytes injected with either water or poly(A)+ mRNA. Values are means ± S.E.M. for 7-10 oocytes per condition, and are representative of two similar experiments; see Materials and methods for details. (B) Dose-dependent expression of D-Phe-L-Ala uptake (N=4). (C) Competition for peptide uptake in oocytes by the simultaneous presence of pairs of peptides (N=4).

View larger version (18K): [in a new window]   Fig. 4. Temperature-dependence of the peptide transporter in BBMV in C. hamatus (A) and A. anguilla (B). The incubation mixture contained 100 mmol l-1 choline chloride (circles) or 100 mmol l-1 choline chloride + 20 mmol l-1 Gly-L-Pro (triangles). For details, see Materials and methods.

View larger version (21K): [in a new window]   Fig. 5. Temperature-dependence of the expression of the peptide transporter in oocytes (diamonds; N=4). Icefish (A) and eel (B) carrier-mediated dipeptide uptake measured in brush-border membrane vesicles (BBMVs; filled circles) is also shown for comparison.