Intestinal absorption of β-lactam antibiotics and oligopeptides : Functional and stereospecific reconstitution of the oligopeptide transport system from rabbit small intestine

Werner KRAMER; Frank GIRBIG; Ulrike GUTJAHR; Simone KOWALEWSKI; Friedhelm ADAM; Werner SCHIEBLER

Febs Journal 204(2): 923-930


ISSN/ISBN: 1742-464X
DOI: 10.1111/j.1432-1033.1992.tb16713.x
Accession: 062242751

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The H+-dependent uptake system responsible for the enteral absorption of oligopeptides and orally active β-lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n-octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active α-amino-cephalosporin, D-cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein-dependent. In these proteoliposomes the binding protein for oligopeptides and β-lactam antibiotics of Mr 127000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles. The uptake system for β-lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D-cephalexin was taken up by intact brush border membrane vesicles, whereas the L-enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127-k Da binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127-k Da protein in the presence of both enantiomers. After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127-k Da binding protein showed a significant uptake of D-cephalexin whereas the L-enantiomer was not transported. The uptake rates for D-cephalexin into proteoliposomes correlated with the content of 127-k Da binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127-k Da binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored. These results indicate that the binding protein for oligopeptides and β-lactam antibiotics of Mr 127000 mediates the stereospecific and H+-dependent transport of orally active β-lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127-k Da binding protein is the intestinal peptide transport system (or a component thereof).