Solubilization and reconstitution characteristics of the carrier protein(s) responsible for the transport of ceftibuten, a substrate for the oligopeptide transporters, in rat renal brush-border membrane

AbstractOptimal procedures for the reconstitution of the transport activity of ceftibuten, a dianionic ß-lactam antibiotic, from rat kidney brush-border membrane were developed. The uptake activity into reconstituted proteoliposomes appeared to be particularly sensitive to the extraction conditions, and to the lipid composition used for reconstitution. Changes in the concentration of octyl glucoside significantly affected the extraction of ceftibuten transport activity, and optimal extraction was achieved at a concentration of 60 mM. Optimal reconstitution was achieved using a lipid composition of asolectin, cholesterol and phosphatidylserine in a w/w percent ratio of 60:30:10, respectively, and with a lipid-to-protein ratio of 10. The uptake of ceftibuten into the resulting proteoliposomes showed temperature and pH dependency, was inhibitable by a range of cephem antibiotics, oligopeptides and the organic anion PAH, and was trans-stimulated by cephalexin and dipeptides. This reconstitution system will likely prove useful in future studies on the functional analysis of the peptide transport system in a purified form