Ligand binding characteristics of two molecular forms, one equipped with a hydrophobic glycosyl phosphatidylinositol tail, of the folate binding protein purified from human milk.
Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [3H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.
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*; D005453:Fluorescence; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008727:Methotrexate / Q000378:metabolism; D008895:Milk, Human / Q000737:chemistry*; D020033:Protein Isoforms; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D011956:Receptors, Cell Surface; D014316:Tritium; D014364:Tryptophan / Q000737:chemistry
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D008024:Ligands; D020033:Protein Isoforms; D011624:Pteroylpolyglutamic Acids; D011956:Receptors, Cell Surface; D014316:Tritium; C036907:pteroylpentaglutamic acid; D014364:Tryptophan; D005492:Folic Acid; D008727:Methotrexate