A Comparison of Copper Uptake by Liver Plasma Membrane Vesicles and Uptake by Isolated Cultured Rat Hepatocytes

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Abstract
We studied copper uptake from copper dihistidine complexes by plasma membrane vesicles isolated from rat liver and compared the data with those for uptake under the same conditions by hepatocytes cultured from rat liver to determine whether membrane vesicles can be used to study copper uptake. Marker enzyme analysis showed a 28–fold increase in 5′–nucleotidase activity, a slight increase in endoplasmic reticulum and no contamination with mitochondrial membranes. Copper uptake by vesicles is temperature dependent, and solubilization with Triton X–100 results in a loss of accumulative capacity. Increasing osmotic pressure resulted in a decrease in copper levels in the vesicles at equilibrium, showing that uptake — as opposed to binding by the vesicles—occurred. Uptake by vesicles is concentration dependent, with evidence for cooperation in the uptake sites. The substrate concentration yielding 10% maximum uptake was 4.01 ± 0.5 μmol/L, maximum uptake was 10.8 ± 0.4 nmol/Cu/mg protein min and the n value was 1.5 ± 0.2. In contrast, uptake by cells showed no cooperation (n = 1.09 ± 0.06) and a significantly higher apparent Michaelis–Menten constant (17.4 ± 1.3 μmol/L). As expected, the maximum uptake was lower in the hepatocytes (1.82 ± 0.08 nmol/mg protein min). Albumin, N –ethylmaleimide and zinc all inhibited uptake in vesicles and in hepatocytes, and the degrees of inhibition were similar in both types of preparation. Vitamin C stimulated uptake in both vesicles and hepatocytes; again, there was a correlation between the increase in uptake at different concentrations. However, cadmium inhibited uptake and nickel stimulated uptake in vesicles and neither metal had any effect in hepatocytes. The data show that vesicles have value as a system in which to study copper uptake but that care must be taken in interpreting the information obtained.(HEPATOLOGY 1994;20:1024–1031)