Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol, phospholipids, and synthetic detergents.
Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.
Holm
Jan
J
Department of Clinical Chemistry, Central Hospital Herning, DK-7400, Denmark. BCMM-JH@BORA.dk
Hansen
Steen Ingemann
SI
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002593:Cetrimonium Compounds / Q000737:chemistry; D002784:Cholesterol / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D008024:Ligands; D008051:Lipid Bilayers; D008892:Milk / Q000737:chemistry; D017830:Octoxynol / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism*; D011136:Polysorbates / Q000737:chemistry; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D002593:Cetrimonium Compounds; D003902:Detergents; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D008051:Lipid Bilayers; D010743:Phospholipids; D011136:Polysorbates; D011956:Receptors, Cell Surface; D017830:Octoxynol; D005492:Folic Acid; D002784:Cholesterol