A PLASMA-MEMBRANE NADH OXIDASE IS INVOLVED IN COPPER UPTAKE BY PLASMA-MEMBRANE VESICLES ISOLATED FROM RAT-LIVER

The accumulation of copper (Cu) by hepatocytes is initiated by the bin ding of Cu in either a CuHis(2) complex or as a CuHisAlb ternary compl ex, followed by transfer of the metal alone across the cell membrane. In this paper, we provide evidence that the transfer involves reductio n of cupric (Cu(II)) copper to cuprous (Cu(I)) copper and further we s how that membrane-bound NADH oxidase can provide the electron required far the reduction. Cu-64 uptake by rat liver plasma membrane vesicles is stimulated by the addition of NADH, but not NAD(+). The stimulatio n increases the V-max from 4.75 +/- 0.02 to 8.38 +/- 0.40 nmol Cu/mg p rotein 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 mu mol/l; with n v alues of 1.30 +/- 0.01 and 1.43 +/- 0.10, respectively; analysing by t he Hill equation). Correspondingly, addition of CuHis(2) stimulated NA DH-oxidase activity by a maximum of 7.4 +/- 2.1 nmol/mg protein per mi n (P < 0.01, mean +/- S.E., n = 5) at 5 mu mol/l and a NADH concentrat ion of 150 mu mol/l. Ascorbic acid also stimulated copper uptake, and points to a reductive dissociation of copper prior to its movement int o 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.