Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling

Abstract

The uptake of a photolabile derivative of the orally effective cephalosporin cephalexin, N-(4-azidobenzoyl)cephalexin, was investigated in brush-border membrane vesicles. The compound was taken up into the intravesicular space and inhibited the active uptake of cephalexin in a concentration-dependent manner. Therefore, this probe interacts with the transport system shared by α-aminocephalosporins and dipeptides. Photoaffinity labeling of brush-border membrane vesicles from rat small intestine with N-(4-azido [3,5,- 3H]benzoyl) derivatives of the cephalosporin cephalexin and the dipeptide glycyl- l-proline resulted in the covalent incorporation of radiaoctivity into membrane polypeptides with apparent molecular weights of 127000, 100000, 94000 and 86000, the polypeptide of molecular weight 127000 being predominantly labeled. The specificity of labeling was demonstrated by a decrease in the labeling of the polypeptide of apparent molecular weight 127000 in the presence of β-lactam antibiotics and dipeptides, whereas glucose, taurocholate or amino acids had not effect on the labeling pattern. These data demonstrate an interaction of cephalosporins and dipeptides with a common membrane protein of molecular weight 127000, which could be a component of the intestinal transport system(s) responsible for the uptake of orally effective cephalosporins and dipeptides.