AJP - Gastrointestinal and Liver Physiology, Vol 251, Issue 4 481-G486, Copyright © 1986 by American Physiological Society
Folate binding and hydrolysis by pig intestinal brush-border membranes
A. M. Reisenauer, C. J. Chandler and C. H. Halsted
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.
