Cu metabolism in the liver.

This paper has, given some idea of our concepts of the processes involved in the transport of Cu across cell membranes in the liver, which we have summarised in Fig 1. Cu(II)His2 is reduced to Cu(I). This is transported across the membrane, re-oxidised, either before or after binding to glutathione (Freedman et al., 1989) or HAH1 (Klomp et al., 1997), binds to SAHH, and donates Cu(II) to the ATPase. It is very interesting that cells which are very diverse from an evolutionary point of view still use very similar methods to handle the metal. Whether regulation of transport is also the sam remains to be seen. We would guess that, although there will be strong similarities, there will also be very significant differences, reflecting the different environments seen by different tissues in mammalian cells and given the different requirements of the tissues.

Physiologic function of the Wilson disease gene product, ATP7B.

The genes responsible for Wilson disease and Menkes syndrome have been cloned and identified as copper ATPases. These enzymes form part of a large family of transporters, the P-type ATPases. Although copper ATPases share strong structural similarities with these other pumps, comparatively little is known about their physiologic function. In this review, we examine data relating to the Wilson disease protein, ATP7B, in the liver. We present evidence suggesting that ATP7B is located intracellularly, together with data suggesting that, at least in part, ATP7B may also be found on the canalicular membrane. We also examine the form of copper that the transporter recognizes. We then review data on the Long-Evans Cinnamon rat, a model for Wilson disease, and discuss what effect the Wilson disease mutation has on copper transport. Finally, we conclude that, although we have made major advances in our understanding of copper metabolism in the liver, there are still many questions awaiting answers.

Characterization of intracellular copper pools in rat hepatocytes using the chelator diamsar.

When hepatocytes are incubated with the chelator diamsar, two pools can be identified, which we have termed extractable and nonextractable. On entering the hepatocyte, 67Cu first associates with the extractable pool and, after approximately 2 h, moves to the nonextractable pool. Both pools demonstrate saturation and are filled as a function of Cu concentration and incubation time. Using the Michaelis-Menten equation, we have estimated the size of the pools after incubation with 67Cu for 30 min and 4 h. During this period the extractable pool decreases in size from 200 +/- 27 to 116 +/- 5 pmol/microgram DNA, whereas the nonextractable pool increases from 28 +/- 9 to 77 +/- 11 pmol/microgram DNA. Movement of Cu from the nonextractable pool to the extractable pool is slow and incomplete. Using [3H]diamsar, we demonstrate that uptake of the chelator is not rate limiting and probably does not occur by pinocytosis. Incubation with diamsar does not affect the activity of superoxide dismutase or cytochrome-c oxidase, although it does prevent the incorporation of 67Cu into ceruloplasmin. Incubation with zinc, which induces metallothionein, results in an increase in 67Cu associated with the nonextractable pool, suggesting that 67Cu-metallothionein constitutes at least part of the nonextractable pool.

ATP-dependent copper transporter, in the Golgi apparatus of rat hepatocytes, transports Cu(II) not Cu(I).

The Wilson disease adenosinetriphosphatase (ATPase; ATP7B) is believed to bind copper as Cu(I). We provide evidence to suggest that the ATPase actually transports Cu as Cu(II). When the copper is presented to rat liver microsomes as Cu(I), virtually all uptake is ATP independent. If the copper is presented as copper oxalate [Cu(II)], total uptake is reduced to approximately 10% of Cu(I) levels, but ATP-dependent uptake rises, both as a proportion of total uptake and in absolute terms. The reducing agent vitamin C and the Cu(I) chelator bathocuproine both override the effect of oxalate. The data indicate that there are two transporters in the microsomes, an ATP-independent Cu(I) transporter and an ATP-dependent Cu(II) pump. The activity of the Cu(I) transporter correlates most strongly with alkaline phosphatase, suggesting that it is derived from plasma membrane contamination. Cu(II) ATP-dependent transport correlates only with beta-1, 4-galactosyltransferase, which indicates that it is located in the Golgi apparatus.

Identification of an ATP-dependent copper transport system in endoplasmic reticulum vesicles isolated from rat liver.

1. This paper identifies and characterizes an ATP-dependent copper transport system in endoplasmic reticulum vesicles isolated from male rat liver. 2. The transporter has a Km of 2.5 +/- 1.2 mumol 1(-1) copper glutathione (CuGSH) and a Vmax of 4.5 +/- 1.3 nmol (mg protein)-1 (5 min)-1 for copper. 3. At a copper concentration of 2 mumol l-1, ATP dependence reaches saturation, with a Km for ATP of 4.7 +/- 2.4 mmol l-1 and a Vmax of 2.8 +/- 0.6 nmol (mg protein)-1 (5 min)-1. 4. The uptake is dependent on ATP hydrolysis, since a low energy analogue of ATP, adenosine 5'-[beta-gamma-methylene] triphosphate tetralithium (AMP.PCP), has no effect on copper uptake. 5. The transporter is a P-type ATPase, since vanadate inhibits uptake with a high degree of specificity (100 mumol l-1 inhibits uptake by 50% at a copper concentration of 2 mumol l-1).

A 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 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 mumol/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 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)