PURIFICATION AND LIPOSOMAL RECONSTITUTION OF THE OLIGOPEPTIDE TRANSPORT ACTIVITY IN RAT RENAL-CORTEX USING CEFTIBUTEN-AFFINITY CHROMATOGRAPHY

The carrier protein(s) responsible for the transport of ceftibuten, a peptide-like dianionic cefem, in rat renal brush-border membrane were solubilized and purified by a ceftibuten-ligand specific affinity chro matography technique. The proteoliposomes reconstituted from the solub ilized brush-border membrane proteins by dialysis had H+-sensitive upt ake of ceftibuten and trans-stimulative effect by cephalexin. A specif ic uptake activity for ceftibuten was found in the 3.5 M-eluted fracti on but not the flowthrough and the 0.5 M-eluted fraction of the affini ty chromatography. Analyzing this active fraction by SDS/PAGE after re constituting into liposomes gave two major proteins (approx. molecular masses of 130 and 107 kDa). The purification protocol presented in th is study permitted an efficient isolation of the carrier proteins resp onsible for the transport of ceftibuten and other peptide-like compoun ds. (C) 1998 Elsevier Science B.V.