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

Abstract

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 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 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 to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. 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-[57Co]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 Km 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.

Absorption and Transport of Cobalamin (Vitamin B12)

“…IN VITRO STUDIES Cobalamin is absorbed in the ileum (17,30), and Herbert (77) suggested that the absorption of cbl was by means of specialized receptors for IF-cbl complex in the ileum. The presence of ileal binding and presumed receptors has been confirmed from various animal species (38,39,70,108). In humans, binding is present over the entire ileum second half of the small intestine.…”

The Intrinsic Factor-Vitamin B12 Receptor and Target of Teratogenic Antibodies Is a Megalin-binding Peripheral Membrane Protein with Homology to Developmental Proteins

“…Cubilin was iodinated (ϳ10 6 Bq/g) by the iodogen method (Pierce). The 125 I-labeled cubilin was purified by S-300 (Pharmacia) gel filtration, and 10 6 cpm was loaded on the megalin column.…”

“…However, early studies have indicated that both an intrinsic factor-B 12 -binding protein (6) and the target protein of teratogenic antibodies (41) could be released, at least in part, from intestinal or renal tissue using mechanical dissociation in the absence of detergents. We further showed that some cubilin was released by heparin, phosphorylethanol- amine, and EDTA, whereas the membrane association was resistant to acid conditions.…”

“…Failure of either IF production or receptor expression leads inevitably to a B 12 deficiency state and disease (2,3). Although the existence of a receptor for IF-B 12 was recognized nearly 3 decades ago (4), its biochemical properties and structure have remained elusive, possibly because of difficulties in purifying it in high amounts from the terminal ileum (5,6). The observation by Seetharam et al (7) that the receptor was expressed in considerably greater amounts in the kidney and yolk sac facilitated the production of antibodies inhibiting IF-B 12 binding (8,9) and confirmed the receptor as a glycoprotein of Ͼ200 kDa.…”

Solubilization of the ileal receptor for intrinsic factor–vitamin B-12 complex by digestion with papain

“…Furthermore, when the availability of Ca2+ is limited, Mg2+ can enhance Ca2+-dependent binding of IF-B-12 complex to its receptor (Beesley & Bacheller, 1980). A number of investigators have utilized mechanical shearing forces (Cotter & Rothenberg, 1976;Okuda & Fujii, 1977) and/or detergent solutions Vol. 200 (Katz & Cooper, 1974;Schneider et al, 1974;Marcoullis & Graisbeck, 1977) to solubilize the ileal IF-B-12-complex receptor from several species.…”