Purification and characterization of folate binding proteins from rat placenta.
Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M(r) of 36,000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50-64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (Ka = 1.6.10(9) l/mol) lower for N5-methyltetrahydrofolate and substantially lower for N5-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.
Department of Medicine, State University of New York Health Science Center at Brooklyn 11203, USA.
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da Costa
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Rothenberg
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D002955:Leucovorin / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D013763:Tetrahydrofolates / Q000378:metabolism / Q000494:pharmacology
D001059:Apoproteins; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; D005492:Folic Acid; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002955:Leucovorin; C005984:5-methyltetrahydrofolate