COMPARISON OF COPPER UPTAKE BY LIVER PLASMA-MEMBRANE VESICLES AND UPTAKE BY ISOLATED CULTURED RAT HEPATOCYTES

We studied copper uptake from copper dihistidine complexes by plasma m embrane vesicles isolated from rat liver and compared the data with th ose 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 vesicl es is temperature dependent, and solubilization with Triton X-100 resu lts in a loss of accumulative capacity. Increasing osmotic pressure re sulted 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 coope ration in the uptake sites. The substrate concentration yielding 10% m aximum uptake was 4.01 +/- 0.5 mu mol/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 sign ificantly higher apparent Michaelis-Menten constant (17.4 +/- 1.3 mu m ol/L). As expected, the maximum uptake was lower in the hepatocytes (1 .82 +/- 0.08 nmol/mg protein.min). Albumin, N-ethylmale-imide and zinc all inhibited uptake in vesicles and in hepatocytes, and the degrees of inhibition were similar in both types of preparation. Vitamin C sti mulated uptake in both vesicles and hepatocytes; again, there was a co rrelation between the increase in uptake at different concentrations. However, cadmium inhibited uptake and nickel stimulated uptake in vesi cles and neither metal had any effect in hepatocytes. The data show th at vesicles have value as a system in which to study copper uptake but that care must be taken in interpreting the information obtained.