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Functional expression of intestinal dipeptide/beta-lactam antibiotic transporter in Xenopus laevis oocytes.

Abstract：

:An intestinal active transport system specific to small peptides and peptide-like drugs such as beta-lactam antibiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal mucosal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H+ co-transporter and is resistant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive beta-lactam antibiotic, [3H]benzylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of beta-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable beta-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.

journal_name

Biochem Pharmacol

journal_title

Biochemical pharmacology

authors

Tamai I,Tomizawa N,Kadowaki A,Terasaki T,Nakayama K,Higashida H,Tsuji A

doi

10.1016/0006-2952(94)90358-1

subject

Has Abstract

pub_date

1994-08-30 00:00:00

pages

881-8

issue

5

eissn

0006-2952

issn

1873-2968

pii

0006-2952(94)90358-1

journal_volume

48

pub_type

杂志文章

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