Solubilization, Partial Purification and Radioassay for the Intrinsic Factor Receptor from the Ileal Mucosa

Abstract

Summary. A macromolecule which binds intrinsic factor saturated with vitamin B₁₂ has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor ‘receptor’was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105 000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105 000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[⁵⁷Co]vitamin-B₁₂ complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[⁵⁷Co]B₁₂ alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [⁵⁷Co]B₁₂. The receptor did not bind intrinsic factor-[⁵⁷Co]B₁₂ below pH 5 while binding was observed topH 9.0. Binding was equivalent at 37° and 25°, but was markedly reduced at 4° and 56° and was destroyed at 100°. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[⁵⁷ Co]B₁₂ to the receptor and this inhibition could be reversed by the addition of excess Ca²⁺. Mg²⁺ and Mn²⁺ were less effective than Ca²⁺ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B₁₂/min with a K_m of 1.36 x 10^-10 M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B₁₂ than for intrinsic factor free of vitamin B₁₂. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.