PURIFICATION BY CEFTIBUTEN-AFFINITY CHROMATOGRAPHY AND THE FUNCTIONALRECONSTITUTION OF OLIGOPEPTIDE TRANSPORTER(S) IN RAT INTESTINAL BRUSH-BORDER MEMBRANE

The transport activity of ceftibuten, a dianionic peptide-like compoun d, was extracted from rat intestinal brush-border membrane by iz-octyl glucoside and reconstituted into asolectin liposomes by dialysis. The proteoliposomes prepared from the membrane extract showed an inward H-gradient-dependent uptake of ceftibuten and glycylsarcosine. Ceftibut en-immobilized affinity chromatography of the membrane extract permitt ed the isolation of two polypeptides (apparent molecular mass of 117 a nd 127 kDa) that can recognize the dianionic peptide structure of ceft ibuten. Proteoliposomes prepared from reconstituting the isolated prot eins into asolectin vesicles showed an overshooting uptake of ceftibut en in the presence of an inwardly directed H+ gradient, and this uptak e could be inhibited by L-valyl-L-proline. N-glycanase digestion of th e isolated proteins, 117 and 127 kDa, trimmed them into 78 and 120 kDa products, respectively. The protein core size of the smaller protein was in agreement with the calculated molecular mass of similar to 79 k Da for the rat PepT1 transporter obtained by other investigators. (C) 1998 Elsevier Science B.V.