High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein.
High-affinity [3H]folate binding in solubilized human choroid plexus homogenate displayed characteristics, e.g. apparent positive co-operativity, which are typical of specific folate binding. The highest folate-binding activity per g of protein was associated with the 27000 g membrane pellet where the membrane-marker enzyme gamma-glutamyltransferase had its main localization. Ultrogel AcA 44 chromatography revealed two major folate-binding proteins (molecular masses greater than 110 kDa and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) in the Triton X-100-solubilized membrane pellet. After exposure of the membrane pellet to phosphatidylinositol-specific phospholipase C there was only one large 25 kDa peak of folate binding. This could suggest that the folate-binding protein is anchored to the membrane by a glycosylphosphatidylinositol moiety, which can be inserted into Triton X-100 micelles and thus can give rise to forms of large molecular size on gel filtration. This notion was supported by the identical molecular masses of the greater than 110 kDa and 25 kDa folate-binding peaks determined by SDS/PAGE and immunoblotting. The folate-binding protein in choroid plexus cross-reacted with rabbit antibodies against the 25 kDa human milk folate-binding protein, and paraffin-embedded sections of choroid plexus showed immunostaining after exposure to rabbit anti-(human milk folate-binding protein) serum (1:8000 dilution).
Holm
J
J
Department of Clinical Chemistry, Central Hospital, Nykøbing Falster, Denmark.
Hansen
S I
SI
Høier-Madsen
M
M
Bostad
L
L
D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002831:Choroid Plexus / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011869:Radioligand Assay; D011956:Receptors, Cell Surface; D010738:Type C Phospholipases / Q000378:metabolism
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase