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Solubilization, Partial Purification and Radioassay for the Intrinsic Factor Receptor from the Ileal Mucosa
Author(s) -
COTTER R.,
ROTHENBERG S. P.
Publication year - 1976
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1976.tb03594.x
Subject(s) - intrinsic factor , chemistry , pronase , chromatography , trypsin , chymotrypsin , receptor , sephadex , centrifugation , size exclusion chromatography , biochemistry , enzyme , stomach
S ummary . A macromolecule which binds intrinsic factor saturated with vitamin B 12 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‐[ 57 Co]vitamin‐B 12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor‐[ 57 Co]B 12 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 [ 57 Co]B 12 . The receptor did not bind intrinsic factor‐[ 57 Co]B 12 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]B 12 to the receptor and this inhibition could be reversed by the addition of excess Ca 2+ . Mg 2+ and Mn 2+ were less effective than Ca 2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B 12 /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 B 12 than for intrinsic factor free of vitamin B 12 . 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.