Kinetic characterization of zinc binding to brush border membranes from rat kidney cortex: interaction with cadmium

Abstract

Extravesicular and intravesicular zinc bindings were evaluated in brush border membrane vesicles isolated from rat kidney cortex. The process was found to be time-, temperature- and substrate concentration-dependent and displayed saturability. Zn 2+ influx measurements revealed a progressive uptake and massive accumulation at equilibrium which was 50 times higher than the amount that could have been accommodated by the intravesicular space calculated from the equilibrium uptake of d-glucose. Initial (5 s) and equilibrium uptakes (2 h) were found not to be osmotically sensitive as modified by adding mannitol to the medium. It was concluded from these results that the uptake involved massive binding of the Zn 2+ to the brush border membranes components. The ionophore A23187 enhanced the rates of uptake and efflux of Zn 2+ without affecting equilibrium values, suggesting binding of Zn 2+ to interior sites of the membranes. Zn 2+ flux measurements led to the conclusion that two vesicular pools of Zn 2+ bindings existed: a small external pool, accessible to cation chelator (EGTA) or competitive cation cadmium and 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 exchangeable ions like zinc and cadmium. Scatchard plot analysis revealed two distinct types of extravesicular binding sites. High affinity extravesicular zinc binding sites reached saturation at 1.6 mM zinc, had a K d of 137 μM and the number of binding sites were 12 nmol/mg protein. Low affinity extravesicular zinc binding sites could not be saturated under experimental conditions up to 3.2 mM zinc. It had a K d of 526 μM and the number of binding sites 28 nmol/mg protein. Interestingly intravesicular binding of zinc revealed only one type of high affinity binding sites ( K d of 104 μM and number of maximal binding sites 400 nmol/mg protein). Furthermore, kinetic analysis of inhibitory effect of Cd 2+ on extravesicular zinc bindings showed an increase in K d of both types of binding sites but there was no significant change in number of maximal binding sites. Extravesicular zinc binding was temperature-sensitive. Arrhenius plot showed the break point at 30°C. The apparent energies of activation were 13.36 Kcal/mol and 3.1 Kcal/mol below and above the break points respectively. The inhibitory effect of sulfhydryl blocking agents on extravesicular zinc binding suggest the involvement of -SH groups in zinc translocation. An increase in initial zinc uptake was observed in the presence of outwardly directed proton gradient. Intravesicular pool of 65Zn was displaced by unlabelled 2 mM Zn 2+ or 2 mM Cd 2+ but not by calcium present in the bathing medium. It is inferred that intravesicular binding sites have a high affinity and are specific for zinc. It is concluded from the present study that in the first instance the binding of zinc to the exofacial zinc binding component and concomitantly its translocation across the membrane, and subsequently massive binding of zinc to interior sites of brush border membranes occurs.