Isolation and characterization of the ileal receptor for intrinsic factor-cobalamin

The receptor for intrinsic factor-cobalamin (vitamin B₁₂) has been purified 65,000-fold from 2.5 kg of canine ileal mucosa with recovery of 26%. The initial purification steps involved solubilization of ileal mucosal homogenates with Triton X-100, followed by ethanol precipitation and dialysis. The soluble receptor was then saturated with hog intrinsic factor-cobalamin and the receptor-hog intrinsic factor-cobalamin complex was adsorbed to anti-hog intrinsic factor-Sepharose. After extensive washing, the receptor was eluted with 5 mM EDTA at pH 5.0 leaving behind the hog intrinsic factor-cobalamin that remained bound to the anti-hog intrinsic factor-Sepharose. Gel filtration on Bio-Gel A-5m with 0.1% Triton X-100 gave two peaks of receptor activity with apparent molecular weights of 7,500,000 and 5,000,000 indicating that the receptor aggregates under these conditions. Polyacrylamide disc gel electrophoresis with 0.1% Triton X-100 gave a single protein band that barely entered 4% gels, did not stain for carbohydrate, and coincided with the presence of intrinsic factor-cobalamin-binding ability. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate gave one major band with an apparent molecular weight of 180,000, which upon reduction with 2-mercaptoethanol gave two bands with apparent molecular weights of 59,000 and 42,000. Amino acid analysis indicated the presence of 55% hydrophobic amino acids, less than 1% amino sugars, and gave a value of 222,000 g of amino acid/mol of intrinsic factor-cobalamin-binding ability. The purified receptor showed specific binding with human, hog, and canine intrinsic factor-cobalamin (K(a) = 1 to 4 nM^-1) but not with free cobalamin, free intrinsic factor, or R protein-cobalamin. Antibodies to the receptor have been raised in rabbits.