Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.
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 degrees C for the former and 25 degrees C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.
Maffia
M
M
Laboratory of General Physiology, Department of Biological and Environmental Science and Technology, University of Lecce, strada prov le Lecce-Monteroni, I-73100 Lecce, Italy. m.mafia@physiology.unile.it
Rizzello
A
A
Acierno
R
R
Verri
T
T
Rollo
M
M
Danieli
A
A
Döring
F
F
Daniel
H
H
Storelli
C
C
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000864:Antarctic Regions; D015820:Cadherins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003081:Cold Climate; D005260:Female; D005768:Gastrointestinal Hormones / Q000502:physiology*; D006454:Hemoglobins / Q000172:deficiency / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000502:physiology*; D007700:Kinetics; D026901:Membrane Transport Proteins; D008871:Microvilli / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010473:Perciformes / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D000596:Amino Acids; C490584:CDH17 protein, human; D015820:Cadherins; D002352:Carrier Proteins; D005768:Gastrointestinal Hormones; D006454:Hemoglobins; D026901:Membrane Transport Proteins; D010455:Peptides; D012333:RNA, Messenger; C067802:intestinal peptide-proton cotransporter