title: Structural studies of the H+/oligopeptide transport system from rabbit small intestine.
abstract: 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 [3H]cephalexin and further photoreactive 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 active alpha-amino-beta-lactam antibiotics (Kramer et al., Eur. J. Biochem. 204 (1992) 923-930). H+-Dependent transport activity was found in all segments of the small intestine concomitantly with the specific labeling of the 127-kDa protein. By enzymatic deglycosylation, fragments of Mr 116 and 95 kDa were obtained from the 127-kDa protein with endoglucosidase F and N-glycanase, whereas with endoglucosidase H, a fragment of Mr 116 kDa was formed. These findings indicate that the photolabeled 127-kDa protein is a microheterogenous glycoprotein. Surprisingly, it was found that the solubilized and purified 127-kDa protein showed enzymatic sucrase and isomaltase activity. Inhibition of the glucosidase activities with the glucosidase inhibitor HOE 120 influenced neither H+/oligopeptide transport nor photoaffinity labeling of the 127-kDa protein. With polyclonal antibodies raised against the purified 127-kDa protein, a coprecipitation of sucrase activity and the photolabeled 127-kDa beta-lactam antibiotic binding protein occurred. Target size analysis revealed a functional molecular mass of 165+/-17 kDa for photoaffinity labeling of the 127-kDa protein, suggesting a homo- or heterodimeric functional structure of the 127-kDa protein in the brush-border membrane. These findings indicate that the H+/oligopeptide binding protein of Mr 127000 is closely associated with the sucrase/isomaltase complex in the enterocyte brush-border membrane.
authors.affiliation: Department of Research on Metabolic Diseases, Hoechst Marion Roussel Deutschland GmbH, D-65926 Frankfurt am Main, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Kramer
authors.forename: F
authors.initials: F
authors.lastname: Girbig
authors.forename: U
authors.initials: U
authors.lastname: Bewersdorf
authors.forename: S
authors.initials: S
authors.lastname: Kohlrautz
authors.forename: C
authors.initials: C
authors.lastname: Weyland
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002506:Cephalexin / Q000737:chemistry; D006031:Glycosylation; D007421:Intestine, Small / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009834:Oligo-1,6-Glucosidase / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D011817:Rabbits; D013393:Sucrase / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D019920:Photoaffinity Labels; D011522:Protons; C067802:intestinal peptide-proton cotransporter; D009834:Oligo-1,6-Glucosidase; D013393:Sucrase; D002506:Cephalexin