Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose.
Intrinsic factor receptor was purified from hog ileum using human intrinsic factor covalently bound to Sepharose. A yield of 49.6% and a specific activity of about 2500 pmol/mg protein were achieved. The purified receptor was very unstable: 24 h of storage or addition of sodium phosphate precipitated it. The association constant of the receptor for the cyano[57Co]cobalamin-intrinsic factor complex was estimated to be 2.1 nM-1. In native polyacrylamide gel electrophoresis it resolved in two 256 and 320 kDa bands; beta-mercaptoethanol treatment cleared it into four bands corresponding to molecular masses of 107, 81.8, 63.5 and 53.2 kDa. An additional 39.3 kDa band was considered to be an artefact due to the presence of Triton X-114. Isoelectric focusing polyacrylamide gel electrophoresis resolved the receptor into two isoproteins isoelectric at pH 4.7 and 5.1. A similar result was obtained in column electrofocusing with the 125I-iodinated receptor. The 125I-labelled receptor did not crossreact with rabbit anti-human intrinsic factor antiserum. The electrophoretic properties of the receptor purified with intrinsic factor covalently bound to Sepharose were compared to those of the receptor purified by the use of the classical cobalamin-affinity medium. It was concluded that a disassembled receptor was produced using the classical method.
Guéant
J L
JL
Minerva Foundation Institute for Medical Research, Helsinki, Finland.
Jokinen
O
O
Schohn
H
H
Monin
B
B
Nicolas
J P
JP
Gräsbeck
R
R
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D005750:Gastric Juice; D006801:Humans; D007082:Ileum / Q000378:metabolism*; D007437:Intrinsic Factor / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018000:Receptors, Peptide; D013552:Swine
D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028199:intrinsic factor receptor; D007437:Intrinsic Factor