INFLUENCE OF AMINO-ACID SIDE-CHAIN MODIFICATION ON THE UPTAKE SYSTEM FOR BETA-LACTAM ANTIBIOTICS AND DIPEPTIDES FROM RABBIT SMALL-INTESTINE

The influence of chemical modification of functional amino acid side-chains in proteins on the H+-dependent uptake system for orally active α-amino-β-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na+-dependent uptake systems for d-glucose and l-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for β-lactam antibiotics also remained unchanged, whereas the uptake of d-glucose and l-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for d-glucose and l-alanine. The involvement of histidine residues in the transport of orally active α-amino-β-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the α-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for β-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127 000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for α-amino-β-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active α-amino-β-lactam antibiotics across the enterocyte brush-border membrane. © 1990.