ZINC-BINDING IN INTESTINAL BRUSH-BORDER MEMBRANE ISOLATED FROM PIG

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). Zn2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160-mu-l/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6-mu-l/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). Zn-65 efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competitive cations, and a large intravesicular pool. Accumulated Zn-65 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(3) M-1, n = 0.455 nmol Zn2+/mg protein) and a second class of extravesicular sites having a very low affinity (K(a) = 22 M-1, n = 25.35 nmol Zn2+/mg protein) and, on the other hand one type of intravesicular sites (K(a) = 3.3.10(4) M-1, n = 550 nmol Zn2+/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 exchange with the internally accumulated labelled cation.