PURIFICATION BY COBALAMIN-SEPHAROSE AFFINITY-CHROMATOGRAPHY AND INTRINSIC FACTOR-BINDING ACTIVITY OF AN EXTRAMEMBRANE PROTEOLYTIC PRODUCT FROM PIG ILEAL MUCOSA

We have purified a cobalamin-binding protein obtained by papain digest ion of pig intestine by cobalamin-AH-Sepharose affinity chromatography , with a purification factor of 17300, a yield of 63% and a cobalamin- binding activity of 11260 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 [Co-57] cobalamin and [57 Co]cobalamin-intrinsic fa ctor in native PAGE autoradiography and it inhibited the binding of in trinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein s hared a binding activity for both cobalamin and intrinsic factor-cobal amin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor.