Characterization of the folate-binding proteins associated with the plasma membrane of rat liver.
Unsaturated folate-binding proteins (i.e., apo forms) have been identified with the plasma membranes of rat liver by the binding of [3H]pteroylglutamic acid. Normal rat liver contains very little of the folate-binding apoproteins, but the folate-binding capacity increases substantially when the rats are made folate-deficient. This increase appears to be due to unsaturation of the folate-binding holoproteins rather than to synthesis of additional protein, because the binding capacity of the plasma membranes from normal rat liver following dissociation of the bound folate is equivalent to the binding capacity of the preparation from folate-deficient liver. Two molecular forms of folate-binding protein were identified by gel filtration of the solubilized plasma membrane fraction, a high-molecular-weight form (Mr less than 100,000), representing 25% of the binding capacity, and a smaller protein (Mr approximately equal to 55,000), representing 75% of the binding capacity. Whereas the larger species can be solubilized only with a detergent, the smaller form appears to be hydrophilic and dissociates spontaneously from the membrane preparation. The binding of [3H]pteroylglutamic acid by the membrane preparation was specific, saturable, and pH- and temperature-dependent. Scatchard analysis of the binding could be fitted to a curvo-linear plot, indicating at least two orders of binding sites which probably correspond to the two molecular forms identified by gel filtration. Competitive inhibition by folate analogues demonstrated that the apoproteins have higher affinity for oxidized folate than for N5-methyltetrahydrofolate and virtually no affinity for N5-formyltetrahydrofolate or methotrexate.
da Costa
M
M
Division of Hematology and Oncology, Brooklyn Veterans Administration, NY.
Rothenberg
S P
SP
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D005494:Folic Acid Deficiency / Q000378:metabolism; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid; D050938:Glycine N-Methyltransferase; C494512:Gnmt protein, rat