Characterization and regulation of a cloned ovine gastrointestinal peptide transporter (oPepT1) expressed in a mammalian cell line

Hong Chen, Xiang Pan Yuan Xiang Pan, E. A. Wong, Jr Webb

Research output: Contribution to journalArticle

Abstract

To investigate the kinetics of peptide transport by the peptide transporter, PepT1, Chinese hamster ovary cells were transfected with an expression vector containing our cloned ovine PepT1 cDNA. Transport was assessed by uptake studies using the radiolabeled dipeptide, [3H]-Gly-Sar. Expression of oPepT1 was detected at 8-24 h post-transfection with an optimal time of 16-24 h. Uptake of Gly-Sar by oPepT1 was pH-dependent with an optimal pH of 5.5-6.0, concentration-dependent and saturable with an apparent Km value of 1.0 ± 0.1 mmol/L and a maximum velocity of 14.3 ± 0.4 nmol/(mg protein·40 min). Competition studies with nonradiolabeled peptides and [3H]-Gly-Sar showed that all di- and tripeptides inhibited uptake of [3H]-Gly-Sar. In addition, three tetrapeptides (Met-Gly-Met-Met, Pro-Phe-Gly-Lys, and Val-Gly-Ser-Glu) also inhibited [3H]-Gly-Sar uptake. There was no inhibition of [3H]-Gly-Sar uptake detected in the presence of nonradiolabeled free amino acids. Treatment of the cells with staurosporine, an inhibitor of protein kinase C (PKC) significantly increased the transport system. This increase was specific and could be blocked if treatment was done in the presence of phorbol 12-myristate-13-acetate (PMA), an activator of PKC. The staurosporine- and PMA-induced changes in peptide transport activity were not affected by cotreatment with cycloheximide. These data demonstrate that the transport of peptide substrates by oPepT1 in transfected mammalian cells is similar to that in microinjected Xenopus oocytes and that PKC phosphorylation plays a regulatory role in oPepT1 function.

Original languageEnglish (US)
Pages (from-to)38-42
Number of pages5
JournalJournal of Nutrition
Volume132
Issue number1
StatePublished - Jan 16 2002
Externally publishedYes

Keywords

  • Activator
  • Cloning
  • Inhibitor
  • Protein kinase
  • Sequence

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Nutrition and Dietetics

Fingerprint Dive into the research topics of 'Characterization and regulation of a cloned ovine gastrointestinal peptide transporter (oPepT1) expressed in a mammalian cell line'. Together they form a unique fingerprint.

  • Cite this