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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References cited
Banks, W. A., Kastin, A. J., Michals, E. A., and Barrera, C. M., Stereospecific transport of Tyr-MIF-1 across the blood-brain barrier by peptide transport system-1.Brain Res. Bull., 25, 589–592 (1990).
Chomcyznski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem., 162, 156–159 (1987).
Friedman, D. I. and Amidon, G. L., Passive and carrier-mediated intestinal absorption components of two angiotensin converting enzyme (ACE) inhibitor prodrugs in rats: Enalapril and fosinopril.Pharm. Res., 6, 1043–1047 (1989a).
Friedman, D. I. and Amidon, G. L., Intestinal absorption mechanism of dipeptide angiotensin converting enzyme inhibitors of the lysyl-proline type: Lisinopril and SQ 29,852.J. Pharm. Sci., 78, 995–998 (1989b).
Ganapathy, V. and Leibach, F. H., Is intestinal peptide transport energized by a proton gradient?Am. J. Physiol., 249, G153-G160 (1985).
Hediger, M. A., Ikeda, T., Coady, M., Gundersen, C. B., and Wright, E. M., Expression of size-selected mRNA encoding the intestinal Na+/glucose contransporter inXenopus laevis oocytes.Proc. Natl. Acad. Sci. USA, 84, 2634–2637 (1987a).
Hediger, M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M., Expression cloning and cDNA sequencing of the Na+/glucose co-transporter,Nature, 330, 379–381 (1987b).
Hu, M. and Amidon, G. L., Passive and carrier-mediated intestinal absorption components of captopril.J. Pharm. Sci., 77, 1007–1011 (1988).
Kato, M., Maegawa, H., Okano, T., Inui, K., and Hori, R., Effect of various chemical modifiers on H+ coupled transport of cephradine via dipeptide carriers in rabbit intestinal brush-border membranes: role of histidine residues.J. Pharmacol. Exp. Ther., 251, 745–749 (1989).
Kimura, T., Transmucosal absorption of small peptide drugs.Pharmacy International, March, 75–78 (1984).
Klingenberg, M., Survey of carrier methodology: strategy for identification, isolation, and characterization of transport systems.Methods in Enzymology, 171, 12–23 (1989).
Kramer, W., Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling.Biochim. Biophys. Acta, 905, 65–74 (1987).
Kramer, W., Girbig, F., Gutjahr, U., Kleemann, H., Leipe, I., Urbach, H. and Wagner, A., Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and β-lactam antibiotics.Biochim. Biophys. Acta, 1027, 25–30 (1990a).
Kramer, W., Dechent, C., Girbig, F., Gutjahr, U. and Neubauer, H., Intestinal uptake of dipeptides and β-lactam antibiotics. I. the intestinal uptake system for dipeptides and β-lactam antibiotics is not part of a brush border membrane peptidase.Biochim. Biophys. Acta, 1030, 41–49 (1990b).
Kramer, W., Girbig, F., Leipe, I. and Petzoldt, E., Direct Photoaffinity labelling of binding proteins for β-lactam antibiotics in rabbit intestinal brush border membranes with [3H]benzylpenicillin.Biochem. Pharmacol., 37, 2427–2435 (1988a).
Kramer, W., Girbig, F., Petzoldt, E., and Leipe, I., Inactivation of the intestinal uptake system for β-lactam antibiotics by diethylpyrocarbonate.Biochim. Biophys. Acta, 943, 288–296 (1988b).
Kramer, W., Leipe, I., Petzoldt, E., and Girbig, F., Characterization of the transport system for β-lactam antibiotics and dipeptides in rat renal brush-border membrane vesicles by photoaffinity labeling.Biochim. Biophys. Acta, 939, 167–172 (1988c).
Lochs, H., Morse, E. L. and Adibi, S. A., Uptake and metabolism of dipeptides by human red blood cells.Biochem. J., 271, 133–137 (1990).
Maniatis, T., Fritsch, E. F., and Sambrook, J., InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.
Matthews, D. M., Absorption of peptides by mammalian intestine. InPeptide Transport in Protein Nutrition (D. M. Matthews and J. W. Payne, eds.), North-Holland, Amsterdam, 1975, pp. 61–146.
Matthews, D. M., Mechanisms of peptide transport.Beitr. Infusionther. Klin. Ernahr., 17, 6–53 (1987).
Miyamoto, Y., Ganapathy, V., and Leibach, F. H., Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter.J. Biol. Chem., 261(32), 16133–16140 (1986).
Miyamoto, Y., Ganapathy, V., Tiruppathi, C., and Leibach, F. H., Involvement of thiol groups in the function of the dipeptide/proton cotransport system in rabbit renal brush-border membrane vesicles.Biochim. Biophys. Acta, 978, 25–31 (1989).
Miyamoto, Y., Thompson, Y. G., Howard, E. F., Ganapathy, V., and Leibach, F. H. Functional expression of the intestinal peptide-proton co-transporter inXenopus oocytes.J. Biol. Chem., 266, 4742–4745 (1991).
Morley, J. S., Hennessey, T. D., and Payne, J. W., Backbone-modified analogues of small peptides: Transport and antibacterial activity.Biochem. Soc. Trans., 11, 798–800 (1983).
Nakashima, E., Tsuji, A., Mizuo, H., and Yamana, T., Kinetics and mechanism of in vitro uptake of amino-β-lactam antibiotics by rat small intestine and relation to the intact-peptide transport system.Biochem. Pharmacol., 33, 3345–3352 (1984).
Okano, T., Inui, K., Maegawa, H., Takano, M., and Hori, R., H+ coupled uphill transport of aminocephalsporins via the dipeptide transport system in rabbit intestinal brush-border membranes.J. Biol. Chem., 261, 14130–14134 (1986a).
Okano, T., Inui, K., Takano, M., and Hori, R., H+ gradient-dependent transport of aminocephalosporins in rat intestinal brush-border membrane vesicles.Biochem. Pharmacol., 35, 1781–1786 (1986b).
Quick, M. W., Naeve, J., Davidson, N., and Lester, H. A., Incubation with horse serum increases viability and decreases background neurotransmitter uptake inXenopus oocytes.Bio Techniques, 13, 358–360 (1992).
Sachs, G. and Fleischer, S., Transport machinery: an overview.Methods in Enzymology, 171, 3–12 (1989).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Oh, DM., Amidon, G.L. & Sadee, W. Functional expressions of endogenous dipeptide transporter and exogenous proton/peptide cotransporter inXenopus oocytes. Arch. Pharm. Res. 18, 12–17 (1995). https://doi.org/10.1007/BF02976500
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02976500