The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells.
Human nasopharyngeal epidermoid carcinoma (KB) cells contain a membrane-associated particulate folate-binding protein which is important in the cellular accumulation of physiologic folates (Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, P. C., and Kolhouse, J. F. (1985) J. Biol. Chem. 260, 14911-14917) and in the binding of methotrexate (Kane, M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) J. Biol. Chem. 261, 44-49). A soluble folate-binding protein appears in media exposed to proliferating KB cells. We have purified to homogeneity both the membrane-associated and the soluble folate-binding proteins from the KB cell tissue culture system. The purified membrane-associated and soluble folate-binding proteins give single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr values of 50,000 and 40,000, respectively. The membrane-associated folate-binding protein contains 45,000 g of amino acids and the soluble folate-binding protein contains 24,000 g of amino acids per mole of folate bound. Each of the purified proteins has a single folate-binding site, and the carbohydrate content is approximately 25% for each species of protein. The affinity constants for 5-methyltetrahydrofolate of the membrane-associated and soluble folate-binding proteins are 0.3 and 2.5 X 10(9) liters/mol, respectively. The affinities of various polyglutamated forms of methotrexate are similar for each protein, increase as the chain length of the polyglutamate increases (from approximately 0.004 X 10(9) liters/mol for methotrexate to 0.3 X 10(9) liters/mol for methotrexate heptaglutamate), are equal to the affinity for 5-methyltetrahydrofolate, and exceed the reported increase in affinity of methotrexate polyglutamates for dihydrofolate reductase.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002294:Carcinoma, Squamous Cell / Q000032:analysis*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007624:KB Cells; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism; D008895:Milk, Human / Q000032:analysis; D008970:Molecular Weight; D009303:Nasopharyngeal Neoplasms / Q000032:analysis*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; C005984:5-methyltetrahydrofolate; D008727:Methotrexate