STRUCTURAL STUDIES OF THE H+ OLIGOPEPTIDE TRANSPORT-SYSTEM FROM RABBIT SMALL-INTESTINE/

A 127-kDa protein was identified as a component of the H+/oligopeptide transport system in brush-border membrane vesicles from rabbit small intestine by photoaffinity labeling with [H-3]cephalexin and further p hotoreactive beta-lactam antibiotics and dipeptides. Reconstitution of stereospecific transport activity revealed the involvement of the 127 -kDa protein in H+-dependent transport of oligopeptides and orally act ive alpha-amino-beta-lactam antibiotics (Kramer et al., Eur. J. Bioche m. 204 (1992) 923-930). H+-Dependent transport activity was found in a ll segments of the small intestine concomitantly with the specific lab eling of the 127-kDa protein. By enzymatic deglycosylation, fragments of M-r 116 and 95 kDa were obtained from the 127-kDa protein with endo glucosidase F and N-glycanase, whereas with endoglucosidase H, a fragm ent of M-r 116 kDa was formed. These findings indicate that the photol abeled 127-kDa protein is a microheterogenous glycoprotein. Surprising ly, it was found that the solubilized and purified 127-kDa protein sho wed enzymatic sucrase and isomaltase activity. Inhibition of the gluco sidase activities with the glucosidase inhibitor HOE 120 influenced ne ither H+/oligopeptide transport nor photoaffinity labeling of the 127- kDa protein. With polyclonal antibodies raised against the purified 12 7-kDa protein, a coprecipitation of sucrase activity and the photolabe led 127-kDa beta-lactam antibiotic binding protein occurred. Target si ze analysis revealed a functional molecular mass of 165+/-17 kDa for p hotoaffinity labeling of the 127-kDa protein, suggesting a homo- or he terodimeric functional structure of the 127-kDa protein in the brush-b order membrane. These findings indicate that the H+/oligopeptide bindi ng protein of M-r 127 000 is closely associated with the sucrase/isoma ltase complex in the enterocyte brush-border membrane. (C) 1998 Elsevi er Science B.V. All rights reserved.