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

Optimal procedures for the reconstitution of the transport activity of ceftibuten, a dianionic beta-lactam antibiotic, from rat kidney brush -border membrane were developed. The uptake activity into reconstitute d proteoliposomes appeared to be particularly sensitive to the extract ion 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 extracti on was achieved at a concentration of 60 mM. Optimal reconstitution wa s achieved using a lipid composition of asolectin, cholesterol and pho sphatidylserine 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 i nhibitable by a range of cephem antibiotics, oligopeptides and the org anic 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 purif ied form.