Zinc binding in intestinal brush-border membrane isolated from pig

Abstract

Zinc binding to brush-border membrane vesicles isolated from pig jejunum was investigated by a rapid filtration method, for long incubation periods (up to 180 min). Zn 2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160 μl/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 μl/mg protein). Changes in medium osmolarity had no effect on zinc accumulation. These results suggested a large degree of zinc binding to vesicular components (membrane or core). 65Zn efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competetive cations, and a large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with an exchange molecule or a chelator. Scatchard plot analyses revealed, on one hand a first class of high-affinity extravesicular zinc binding sites ( K a = 8.6 · 10 3M −1, n = 0.455nmol Zn 2+/mg protein) and a second class of extravesicular sites having a very low affinity ( K a = 22M −1, n = 25.35nmol Zn 2+/mg protein) and, on the other hand one type of intravesicular sites ( K a = 3.3 · 10 4M −1, n = 550 nmol Zn 2+/mg protein). The intravesicular sites have a high affinity for zinc and are specific, since only nonlabelled zinc (or cadmium) but not calcium present in the bathing medium is exchanged with the internally accumulated labelled cation.