Ligand binding and polymerization characteristics of human milk folate binding protein depend on concentration of purified protein and presence of amphiphatic substances.
Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.
Holm
Jan
J
Department of Clinical Chemistry, Central Hospital Herning, DK-7400, Denmark. BCMM-JH@BORA.dk
Hansen
Steen Ingemann
SI
D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D002850:Chromatography, Gel; D003956:Dialysis; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008894:Milk Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008895:Milk, Human / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D011108:Polymers; D011485:Protein Binding; D011869:Radioligand Assay; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D002593:Cetrimonium Compounds; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D008894:Milk Proteins; D010713:Phosphatidylcholines; D011108:Polymers; D011956:Receptors, Cell Surface; D017830:Octoxynol; D005492:Folic Acid; D000077286:Cetrimonium