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 alpha-amino-beta-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 beta-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 alpha-amino-beta-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 alpha-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 beta-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 alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.
Kramer
W
W
Hoechst Aktiengesellschaft, Frankfurt am Main, F.R.G.
Dürckheimer
W
W
Girbig
F
F
Gutjahr
U
U
Leipe
I
I
Oekonomopulos
R
R
D000345:Affinity Labels / Q000378:metabolism; D000409:Alanine / Q000493:pharmacokinetics; D000818:Animals; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics*; D001692:Biological Transport; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004151:Dipeptides / Q000493:pharmacokinetics*; D005947:Glucose / Q000493:pharmacokinetics; D006639:Histidine; D007421:Intestine, Small / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship; D014443:Tyrosine; D047090:beta-Lactams
D000345:Affinity Labels; D000900:Anti-Bacterial Agents; D004151:Dipeptides; D047090:beta-Lactams; D014443:Tyrosine; D006639:Histidine; D005947:Glucose; D004047:Diethyl Pyrocarbonate; D000409:Alanine