FUNCTIONAL EXPRESSION OF INTESTINAL DIPEPTIDE BETA-LACTAM ANTIBIOTIC TRANSPORTER IN XENOPUS-LAEVIS OOCYTES

An intestinal active transport system specific to small peptides and p eptide-like drugs such as beta-lactam antibiotics was functionally exp ressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal mucosal cells. The transport activ ity was evaluated by measuring the uptake of a tripeptide-like cephalo sporin antibiotic, ceftibuten, which has high affinity for the intesti nal peptide/H+ co-transporter and is resistant to peptidases. Ceftibut en transport in mRNA-injected oocytes was pH dependent (a proton gradi ent 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 s howed cis-inhibitory and trans-stimulatory effects on the uptake of ce ftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipe ptides 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 inte stinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the memb rane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive beta-lactam antibiotic, [H-3 ]benzylpenicillin followed by SDS-PAGE analysis of the radiolabeled pr otein. Furthermore, an experiment with mRNA size-fractionated by sucro se density gradient centrifugation indicated that the peptide transpor ter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtai ned using a heterologous gene expression technique, confirm that intes tinal absorption of beta-lactam antibiotics occurs through a carrier-m ediated mechanism and show that biologically stable beta-lactam antibi otics can be useful probes for molecular analysis of intestinal peptid e transporter.