Affinity labelling of the folate-binding protein in pig intestine

Abstract

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.