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Archives of Pharmacal Research

, Volume 18, Issue 1, pp 12–17 | Cite as

Functional expressions of endogenous dipeptide transporter and exogenous proton/peptide cotransporter inXenopus oocytes

  • Doo-Man Oh
  • Gordon L. Amidon
  • Wolfgang Sadee
Research Articles

Abstract

It is essential to clone the peptide transporter in order to obtain better understanding of its molecular structure, regulation, and substrate specificity. Characteristics of an endogenous peptide transporter in oocytes were studied along with expression of an exogenous proton/peptide cotransporter from rabbit intestine. And further efforts toward cloning the transporter were performed. The presence of an endogenous peptide transporter was detected inXenopus laevis oocytes by measuring the uptake of 0.25 μM (10Ci/ml) [3H]-glycylsarcosine (Gly-Sar) at pH 5.5 with or without inhibitors. Uptake of Gly-Sar in oocytes was significantly inhibited by 25 mM Ala-Ala, Gly-Gly, and Gly-Sar (p<0.05), but not by 2.5 mM of Glu-Glu, Ala-Ala, Gly-Gly, Gly-Sar and 25 mM glycine and sarcosine. This result suggests that a selective transporter is involved in the endogenous uptake of dipeptides. Collagenase treatment of oocytes used to strip oocytes from ovarian follicles did not affect the Gly-Sar uptake. Changing pH from 5.5 to 7.5 did not affect the Gly-Sar uptake significantly, suggesting no dependence of the endogenous transporter on a transmembrane proton gradient. An exogenous H+/peptide cotransporter was expressed after microinjection of polyadenylated messenger ribonucleic acid [poly(A)+-mRNA] obtained from rabbit small intestine. The Gly-Sar uptake in mRNA-injected oocytes was 9 times higher than that in water-injected oocytes. Thus, frog oocytes can be utilized for expression cloning of the genes encoding intestinal H+/peptide cotransporters. Size fractionation of mRNA was successfully obtained using this technique.

Key words

Cotransporter Dipeptide Expression Glycylsarcosine Oocytes 

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Copyright information

© The Pharmaceutical Society of Korea 1995

Authors and Affiliations

  • Doo-Man Oh
    • 1
    • 2
  • Gordon L. Amidon
    • 3
  • Wolfgang Sadee
    • 2
  1. 1.College of PharmacyHyosung Women’s UniversityKyoungbookKorea
  2. 2.Department of Pharmacy and Pharmaceutical ChemistryUniversity of CaliforniaSan FranciscoU.S.A.
  3. 3.College of PharmacyThe University of MichiganAnn ArborU.S.A.

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