Affinity labelling of the folate-binding protein in pig intestine.
A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.
Reisenauer
A M
AM
Department of Internal Medicine, University of California Davis 95616.
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000648:ultrastructure; D008727:Methotrexate / Q000031:analogs & derivatives / Q000494:pharmacology; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D011956:Receptors, Cell Surface; D013388:Succinimides / Q000494:pharmacology*; D013552:Swine
D000345:Affinity Labels; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013388:Succinimides; C039702:methotrexate N-hydroxysuccinimide ester; D005492:Folic Acid; D008727:Methotrexate