A plasma membrane NADH oxidase is involved in copper uptake by plasma membrane vesicles isolated from rat liver

Abstract

The accumulation of copper (Cu) by hepatocytes is initiated by the binding of Cu in either a CuHis 2 complex or as a CuHisAlb ternary complex, followed by transfer of the metal alone across the cell membrane. In this paper, we provide evidence that the transfer involves reduction of cupric (Cu(II)) copper to cuprous (Cu(I)) copper and further we show that membrane-bound NADH oxidase can provide the electron required for the reduction. 64Cu uptake by rat liver plasma membrane vesicles is stimulated by the addition of NADH, but not NAD +. The stimulation increases the V max from 4.75 ± 0.02 to 8.38 ± 0.40 nmol Cu/mg protein per min ( P < 0.05, mean ± S.E., n = 3 ) without significantly altering the K 0.5 ( 1.52 ± 0.17 and 2.10 ± 0.22 μ mol/1 ; with n values of 1.30 ± 0.01 and 1.43 ± 0.10 , respectively; analysing by the Hill equation). Correspondingly, addition of CuHis 2 stimulated NADH-oxidase activity by a maximum of 7.4 ± 2.1 nmol/mg protein per min ( P < 0.01, mean ± S.E., n = 5 ) at 5 μmol/1 and a NADH concentration of 150 μmol/1. Ascorbic acid also stimulated copper uptake, and points to a reductive dissociation of copper prior to its movement into the cell. Our data indicate that membrane bound enzymes can provide an electron for the reduction of copper prior to uptake and suggest a physiological role for the plasma membrane NADH oxidase.