Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.
A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.
Guéant
J L
JL
Group of Biochemistry-Immunology, INSERM U 308 University of Nancy I, Vandoeuvre-lès-Nancy, France.
Schohn
H
H
Brulé
H
H
Saunier
M
M
Gräsbeck
R
R
Nicolas
J P
JP
D000327:Adsorption; D000818:Animals; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003037:Cobalt Radioisotopes; D007082:Ileum / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007437:Intrinsic Factor / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide; D015203:Reproducibility of Results; D012685:Sepharose / Q000031:analogs & derivatives; D013552:Swine
D003037:Cobalt Radioisotopes; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028199:intrinsic factor receptor; C020927:phenyl-sepharose; D007437:Intrinsic Factor; D012685:Sepharose