Folate binding and hydrolysis by pig intestinal brush-border membranes

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Abstract
The intestinal absorption of dietary polyglutamyl folate involves hydrolysis, binding, and transport at the brush-border surface of the enterocyte. We studied the binding of [3H]folic acid ([3H]PteGlu) and the hydrolysis of [14C]pteroyltriglutamate ([14C]PteGlu3) by use of brush-border vesicles prepared from pig jejunal mucosa. [3H] PteGlu associated with the vesicles was not affected by increasing the osmolarity of the incubation solution, verifying that the experiments describe binding and not transport of PteGlu. The binding of [3H]PteGlu was saturable (Kd = 0.08 microM) and pH dependent with maximal binding at pH 5.2. Binding was competitively inhibited by PteGlu3 (Ki = 0.25 microM) and 5-methyltetrahydrofolate (Ki = 0.8 microM) but was not affected by components of the PteGlu molecule. ZnCl2, MgCl2, and MnCl2 enhanced the binding capacity but not the affinity of the binding component for PteGlu. We distinguished the processes of folate binding and hydrolysis by demonstrating differences in metal ion requirements and susceptibilities to various inhibitors. In addition, the binding component and the hydrolytic enzyme had distinct affinities for PteGlu (Ki = 45 microM for PteGlu3 hydrolysis). These data demonstrate pH-dependent, specific, and saturable binding of PteGlu to the intestinal brush-border membrane and suggest that the binding component is separate from the hydrolytic enzyme.