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 microliters/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 microliters/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 competitive 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 (Ka = 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 (Ka = 22 M-1, n = 25.35 nmol Zn2+/mg protein) and, on the other hand one type of intravesicular sites (Ka = 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 exchanged with the internally accumulated labelled cation.
Tacnet
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Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
Watkins
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DW
Ripoche
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P
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007408:Intestinal Absorption; D007583:Jejunum / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D009994:Osmolar Concentration; D013012:Sorbitol / Q000378:metabolism; D013552:Swine; D015032:Zinc / Q000378:metabolism*
D002104:Cadmium; D000001:Calcimycin; D013012:Sorbitol; D004533:Egtazic Acid; D015032:Zinc; D002118:Calcium