Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa

• Published in: Biochemical journal Vol. 313 ( Pt 2); no. 2; pp. 675 - 681
• Main Authors: Yerima, A, Safi, A, Gastin, I, Michalski, J C, Saunier, M, Gueant, J L
• Format: Journal Article
• Language: English
• Published: England 15.01.1996
• Subjects: Animals; Autoradiography; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Ileum - metabolism; Intestinal Mucosa - metabolism; Intrinsic Factor - isolation & purification; Intrinsic Factor - metabolism; Kinetics; Protein Binding; Proteins - isolation & purification; Proteins - metabolism; Swine; Vitamin B 12 - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3130675

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.