Intrinsic factor covalently bound to Sepharose as affinity medium for the purification of a soluble intrinsic factor receptor from human urine.
We have identified a soluble receptor for intrinsic factor (IF) in human urine. The purification of this protein by affinity chromatography required a preliminary purification of IF from hog pyloric mucosal extract. This was achieved by thermolabile cobalamin-ethanol-aminohexane Sepharose affinity chromatography with a 133-fold purification, a yield of 45% and a specific binding activity of 15720 pmol/mg protein. The purified Cbl-IF complex was coupled to epoxy-Sepharose with a yield of 23.8% and a specific activity of 1.2 nmol per mol of gel. The soluble IF receptor was purified form 200 ml of urine concentrate of pregnant women. Desorption was performed at pH 5.0 and in the presence of 5 mM EDTA. The soluble IF receptor was purified 17,200-fold with a yield of 52% and a IF binding capacity of 3260 pmol per mg of protein. A single protein with a Mr of 70,000 was found in silver-stained SDS-PAGE.
Safi
A
A
Laboratory of Cellular and Molecular Biology in Nutrition, Medical Faculty, University of Nancy, France.
Saunier
M
M
Gastin
I
I
Alibada
Y
Y
Dugue
B
B
Gueant
J L
JL
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D005753:Gastric Mucosa / Q000378:metabolism; D006801:Humans; D007437:Intrinsic Factor / Q000302:isolation & purification / Q000378:metabolism*; D011247:Pregnancy / Q000652:urine; D011485:Protein Binding; D018000:Receptors, Peptide / Q000302:isolation & purification*; D012685:Sepharose / Q000378:metabolism*; D013552:Swine
D018000:Receptors, Peptide; C028199:intrinsic factor receptor; D007437:Intrinsic Factor; D012685:Sepharose