PURIFICATION AND CHARACTERIZATION OF FOLATE BINDING-PROTEINS FROM RATPLACENTA

Rat placenta contains virtually no unsaturated (i.e., ape-form) folate binding protein. However, by lowering the pH of a solubilized membran e preparation of this tissue to 3.5, the endogenous bound folate was d issociated from the protein and adsorbed to charcoal. The ape-form of the folate binding protein thus obtained was purified by affinity chro matography 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 affi nity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa prote in band, However, peptide sequencing of the N-terminal region of the p roteins in the affinity eluate established that it contained two homol ogous proteins. Computer alignment of the first 22 N-terminal amino ac ids of each rat placental protein with human, bovine milk and mouse fo late binding proteins showed 50-64% identical homology and 27% homolog y when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (K-a = 1.6 . 10(9) l/ mol) lower for N-5-methyltetrahydrofolate and substantially lower for N-5-formyltetrahydrofolate. In the dose-response range studied there w as no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phos pholipase C indicating that these proteins belong to the class of prot eins anchored to the plasma membrane by a glycosyl phosphatidylinosito l adduct.