title: Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.
abstract: We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor.
authors.affiliation: Laboratoire de Biologie Cellulaire et Moléculaire en Nutrition et INSERM Unité 308 Faculté de Médecine, Université de Nancy, France.
authors.forename: A
authors.initials: A
authors.lastname: Yerima
authors.forename: A
authors.initials: A
authors.lastname: Safi
authors.forename: I
authors.initials: I
authors.lastname: Gastin
authors.forename: J C
authors.initials: JC
authors.lastname: Michalski
authors.forename: M
authors.initials: M
authors.lastname: Saunier
authors.forename: J L
authors.initials: JL
authors.lastname: Gueant
mesh_terms: D000818:Animals; D001345:Autoradiography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007082:Ileum / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007437:Intrinsic Factor / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D014805:Vitamin B 12 / Q000378:metabolism*
chemical_list: D011506:Proteins; D007437:Intrinsic Factor; D014805:Vitamin B 12