Copper transport and cytosolic distribution in normocupric and Cu-loaded fibroblasts

Copper transport kinetics examined in fibroblasts to obtain the kinetic parameters for an extrahepatic cell. Initial velocity (30s) data gave Km (5.7 +/- 0.8 μM) and Vmax (2.2 +/- 0.2 nmol min-1 mg-1) which were comparable to the parameters for hepatocytes. Copper uptake was also detected with His2 Cu (Km 10.3 +/- 0.2 μM) which was proposed as the plasma species that delivers Cu to hepatocytes. Albumin (2 μM) markedly inhibited Cu-uptake by complexing Cu and zinc competitively inhibited Cu-uptake. Fibroblasts were preincubated with 0.4 mM CuSO4 for 48h to determine the effects of increased metallothionein on Cu-transport. Cu-preloading had no significant effect on the initial velocities of Cu-uptake (Km 11.0 +/- 1.3 μM, Vmax 1.9 +/- 0.1 nmol min-1 mg-1). Sephadex G150 profiles of the cytosolic distribution of newly-arrived 64Cu (15 min) showed three Cu-binding fractions (Mr >300 Kd, ≅ 88Kd and ≅ 10 Kd). A 38 Kd Cu-binding component(s) that was detected with hepatocytes was not detected with fibroblasts. Thus, fibroblasts seem to contain the same Cu-transport system as hepatocytes, and can obtain Cu from His2 Cu. At the levels obtained in these experiments, increased metallothionein did not effect Cu-transport. The cytosolic distribution of newly-arrived Cu exhibits tissue-specificity