title: Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles.
abstract: L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.
authors.affiliation: Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
authors.forename: C
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: P
authors.initials: P
authors.lastname: Kulanthaivel
authors.forename: V
authors.initials: V
authors.lastname: Ganapathy
authors.forename: F H
authors.initials: FH
authors.lastname: Leibach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D007668:Kidney / Q000648:ultrastructure*; D007700:Kinetics; D008564:Membrane Potentials; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009550:Nigericin / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011522:Protons; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D011522:Protons; D007531:Isoflurophate; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D012964:Sodium; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D009550:Nigericin; D011188:Potassium