Characterisation of the H+/peptide cotransporter of eel intestinal brush-border membranes

H+/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel ( Anguilla anguilla) intestine was studied by measuring D-[H-3]-phenylalanyl- L-alanine uptake and by monitoring peptide-dependent intravesicular acidifi cation using the pH-sensitive dye Acridine Orange, D-[H-3]-phenylalanyl-L-a lanine influx was greatly stimulated by an inside-negative membrane potenti al and enhanced by an inwardly directed H+ gradient. In parallel, vesicular Hf influx was significantly increased in the presence of extravesicular D- phenylalanyl-L-alanine or a series of glycyl and L-prolyl peptides. H+/pept ide cotransport displayed saturable kinetics involving a single carrier sys tem with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on t he particular peptide. All substrates tested competed with this system. Pre -incubation of BBMVs,vith dipeptides prevented diethylpyrocarbonate inhibit ion of transport activity, suggesting that the substrates mask histidine re sidues involved in the catalytic function of the transporter. Using human P epT1-specific primers, a reverse transcriptisn-polymerase chain reaction (R T-PCR) signal was detected in eel intestine. Our results suggest that, in e el intestine, a brush-border membrane 'low-affinity'-type H+/peptide cotran sport system is present that shares kinetic features with the mammalian int estinal PepT1-type transporters.