Folate transport in isolated brush border membrane vesicles from rat intestine

The transport of folates by the intact intestine consists of a saturable component with a luminal pH optimum of 6.0 and a nonsaturable component which predominates at high folate concentrations and/or high luminal pH. We sought to determine whether these properties reflect the behavior of the brush border membrane, free of intracellular enzyme activities, organelles, and intercellular junctions. We have studied, therefore the transport characteristics of folic acid (PteGlu) and methotrexate in isolated brush border membrane vesicles from rat intestine. Both PteGlu and methotrexate were found to be taken up by these vesicles by a pH-dependent process with a maximum uptake at a medium pH near 5.0. Studies at pH 5.5 demonstrated both saturable and nonsaturable components for the uptake of methotrexate and PteGlu. For methotrexate, Km, corrected for the nonsaturable component, was 1.5 microM and Vmax = 3.08 pmol/mg of protein/0.5 min. For PteGlu, the Km was 0.42 microM and Vmax = 0.67 pmol/mg of protein/0.5 min. Methotrexate uptake was competitively inhibited by PteGlu (Ki = 0.6 microM and by 5-methyltetrahydropteroylglutamic acid (5-methyl-H4PteGlu; Ki = 1.35 microM). Brush border membrane vesicles exhibit folate transstimulation: those preloaded with PteGlu, 5-methyl-H4PteGlu or unlabeled methotrexate took up tritium labeled methotrexate at more rapid rates than did control vesicles. The data presented are consistent with a pH-dependent, structure-specific carrier or channel for folate transport across the luminal membrane of the intestinal cell shared by the three folate derivatives tested.