Transport of peptides in renal brush border membrane vesicles. Suitability of 125I-labelled tyrosyl peptides as substrates.
Three tyrosine-containing peptides (Tyr-Pro, Tyr-Pro-Phe and Tyr-Pro-Phe-Pro) were [125I]iodo-labelled and their uptake characteristics in renal brush border membrane vesicles was investigated to assess the suitability of these peptides as substrates in peptide transport studies. Hydrolysis of these peptides during uptake measurements was avoided by using brush border membrane vesicles prepared from the kidneys of Japan Fisher 344 rats which genetically lack dipeptidylpeptidase IV activity. The uptake of 125I-Tyr-Pro and 125I-Tyr-Pro-Phe in these membrane vesicles was found to be stimulated by an inwardly directed H+ gradient. In both cases, the time course of uptake exhibited an overshoot, an indication for active transport. The uptake processes were electrogenic since they were stimulated by an inside-negative membrane potential. Free amino acids had no effect on the uptake of Tyr-Pro and Tyr-Pro-Phe whereas many di- and tripeptides effectively blocked the uptake. These results demonstrated that the [125I]iodo-labelled Tyr-Pro and Tyr-Pro-Phe are suitable substrates for peptide transport studies because their uptake characteristics clearly suggest participation of the peptide transporter in their uptake. In contrast, the uptake of 125I-Tyr-Pro-Phe-Pro was not active, and was not stimulated by the H+ gradient or by the membrane potential. In addition, competition experiments with unlabelled amino acids and peptides indicated that the uptake did not occur via the peptide transporter. However, when allowed to be hydrolyzed to 125I-Tyr-Pro and Phe-Pro by using dipeptidylpeptidase IV-positive renal brush border membrane vesicles prepared from USA Fisher 344 rats, the radiolabel from the [125I]iodo-labelled tetrapeptide was found to be taken up into the vesicles via the peptide transporter. These data provide direct evidence to show that intact tetrapeptides are not substrates for the renal peptide transporter.
Tiruppathi
C
C
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Ganapathy
V
V
Leibach
F H
FH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D007457:Iodine Radioisotopes; D007668:Kidney / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D008564:Membrane Potentials; D008871:Microvilli / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D011522:Protons; D051381:Rats; D011916:Rats, Inbred F344; D014443:Tyrosine / Q000378:metabolism*
D000596:Amino Acids; D007457:Iodine Radioisotopes; D010455:Peptides; D011522:Protons; D014443:Tyrosine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4