The Accumulation of Copper by Microvillar Vesicles Isolated from Human Placenta

Abstract

Copper uptake from copper-dihistidine complexes by microvillar vesicles from human placenta was studied. Uptake occurred in two phases: a rapid initial binding followed by approximately linear uptake to equilibrium at ∼5 min. The uptake showed temperature dependence, was saturable (apparent Vmax 10.5 ± 1.6 nmol/(mg protein·4 min), apparent Km of 0.6 ± 0.12 μmol/L) and decreased with increasing osmotic pressure, showing that the Cu uptake arose from accumulation within the vesicles and not from extravesicular binding or isotope exchange. Ceruloplasmin blocked uptake of 64Cu from 64Cu-dihistidine by the vesicles, with 50% inhibition achieved at a protein concentration of 5–10 μmol/L and a 64Cu-dihistidine concentration of 1.5 μmol/L. The effect was specific, because glucose oxidase, a noncopper protein, increased apparent uptake by binding copper and in turn being bound to the nitrocellulose membranes used to separate vesicles from incubation medium. Adding increasing concentrations of histidine also decreased uptake. The data presented indicate that the placenta can accumulate copper from copper-dihistidine, that ceruloplasmin can interfere with uptake and that this system will be very valuable in elucidating the first stage in transfer of copper across the placenta.