Metallothionein-bound copper

Copper The daily intake from food is 0.8—2.0 mg it is released into the portal vein via copper-transporting ATPase. The transport of copper, which is toxic in its free form, is effected by the binding to ceruloplasmin, albumin and transcuprin. Copper is bound to reduced glutathione and metallothionein in the hepatocytes and distributed to various organelles or incorporated into enzymes. The biological effects of copper are manifold and essential for some cellular functions, (s. p. 50) Copper is toxic not only in its free form, but also in cases of overload (e. g. cirrhosis in childhood due to the consumption of water from copper pipes). Copper homoe-ostasis is regulated via biliary excretion (normal value about 1.2-2.0 mg/day), so that the normal value in serum is 75-130 fg/dl. (321, 323, 370, 383, 386) (s. p. 102)... 

If one had to state an overall role of copper in the body, one might say oxygen metabolism. One major factor shared by both zinc and copper is that both metal ions occur bound to metallothionein. The function of metallothionein is not firmly established. Copper is bound to another protein, ceruloplasmin, which occurs in the cell and plasma. The function of this protein is not clear either. Zinc absorption, as iron absorption, is impaired by high levels of phytic acid. Copper absorption is not inhibited by phytic acid. The major route of excretion of both metal ions is fecal, rather than urinary. 

In affected cells, copper significantly accumulates as metallothionein-bound copper in the cytosol and copper transport to the organelles - as well as copper efflux - is disturbed. As a result, cuproenzymes... 

In the liver a substantial amount of copper is metallothionein bound, in contrast to the traces found in kidney tissue. In these experiments a Zn/Cd/Cu ratio of 9/8/3 was observed. 

The basic defect in this disease appears to be in the interaction of copper with the carrier protein caeruloplasmin to which it normally binds strongly. In many of the victims caeruloplasmin levels are low and an abnormally large amount of copper is weakly bound to serum albumin. However, some disease-free heterozygotes also have low levels of caeruloplasmin, while a minority of homozygotes have both normal caeruloplasmin levels and Wilson s Disease. The loss of copper in bile is often lower than normal and a metallothionein-like copper protein may be induced in the liver, perhaps causing the copper retention. An increased copper uptake firom the diet may also occur. Renal damage arises in the later stages of Wilson s disease, perhaps caused in part by the accumulation of a copper-metallothionein. 

The hepatic uptake of diet-derived copper occurs via the copper transporter 1 (Ctrl), which transports copper with high affinity in a metal-specific, saturable fashion at the hepatocyte plasma membrane (Lee et al., 2001 Klomp et al., 2002). After uptake copper is bound to metallothionein (MT), a cytosolic, low molecular weight, cystein-rich, metal binding protein. MT I and MT II are ubiquitously expressed in all cell types including hepatocytes, and have a critical role to protect intracellular proteins from copper toxicity (Palmiter, 1998 Kelley and Palmiter, 1996). The copper stored in metallothionein can be donated to other proteins. Specific pathways allow the intracellular trafficking and compartmentaUzation of copper, ensuring adequate cuproprotein synthesis while avoiding cellular toxicity (Fig.21.1). 

Metal metabolism The use of zinc in the treatment of copper deposition due to Wilson s disease was first proposed in the Netherlands by Schouwink in his 1961 PhD thesis [107, 108 ]. This was based on earher experience in veterinary medicine in Australia that zinc salts are effective in copper poisoning in sheep. Later observations showed that stimulation by zinc of metallothionein blocks the intestinal absorption of copper. Metallothionein binds both zinc and copper, but it has a higher affinity for the latter. It binds newly absorbed copper in enterocytes and prevents it from passing firom the gut into the circulation. Copper has no enterohepatic circulation, and Ihe shedding of enterocytes with copper still bound to metallothionein results in higher fecal copper excretion. 

Gold (Au), when administered to the rat as either NaAuCl4, or sodium aurothiomalate, accumulates in the particulate component of the kidney and in low molecular weight metalloprotein fraction of the cytosol. Induction of metallothionein synthesis by Au not only seems to be less efficient than that in response to mercury (Hg), cadmium (Cd) and bismuth (Bi) (PiOTROWSKi et al. 1979), but also to be determined by changes in copper distribution (Mo-GiLNiCKA and Webb 1981). Pre-treatment with Cd increased the contents of metallothionein-bound Au, Cu and Zn in the hamster kidney (Mogilnicka and Webb 1982). 

Physiologically important metal ions include iron, copper, cobalt and nickel. Normally, metal ions are not present in free solution to any significant extent, but are bound to transport proteins (in plasma) or storage proteins and enzymes (in cells). Thus, iron is bound to transferrin (in plasma) and haemosiderin and ferritin in tissues, copper is bound to caeruloplasmin in plasma, and metallothionein in plasma binds a wide variety of metal ions. The adverse effects of iron overload (section 4.5.1) are the result of free iron, not bound to storage proteins, acting as a source of oxygen radicals. 

In mammals, as in yeast, several different metallothionein isoforms are known, each with a particular tissue distribution (Vasak and Hasler, 2000). Their synthesis is regulated at the level of transcription not only by copper (as well as the other divalent metal ions cadmium, mercury and zinc) but also by hormones, notably steroid hormones, that affect cellular differentiation. Intracellular copper accumulates in metallothionein in copper overload diseases, such as Wilson s disease, forming two distinct molecular forms one with 12 Cu(I) equivalents bound, in which all 20 thiolate ligands of the protein participate in metal binding the other with eight Cu(I)/ metallothionein a molecules, with between 12-14 cysteines involved in Cu(I) coordination (Pountney et ah, 1994). Although the role of specific metallothionein isoforms in zinc homeostasis and apoptosis is established, its primary function in copper metabolism remains enigmatic (Vasak and Hasler, 2000). 

Metallothioneins (MT) are unique 7-kDa proteins containing 20 cysteine molecules bounded to seven zinc atoms, which form two clusters with bridging or terminal cysteine thiolates. A main function of MT is to serve as a source for the distribution of zinc in cells, and this function is connected with the MT redox activity, which is responsible for the regulation of binding and release of zinc. It has been shown that the release of zinc is stimulated by MT oxidation in the reaction with glutathione disulfide or other biological disulfides [334]. MT redox properties led to a suggestion that MT may possesses antioxidant activity. The mechanism of MT antioxidant activity is of a special interest in connection with the possible antioxidant effects of zinc. (Zinc can be substituted in MT by some other metals such as copper or cadmium, but Ca MT and Cu MT exhibit manly prooxidant activity.)... 

Foetal and neonatal livers contain exceptionally high levels of copper compared to the adult organ. Thus, the livers of new-born rats contain as much as 20 times the level of copper and zinc metallothionein as that found in 70-day-old rats.1150 Again, a metallothionein from foetal bovine liver contained eight copper and two zinc atoms per molecule of protein.1151 These proteins can only be isolated with difficulty under oxygen-free conditions. It appears then that large amounts of copper (and zinc) are stored in the liver bound to metallothionein, and are mobilized as required for enzyme synthesis after birth. 

The primary organs for copper storage are the liver and spleen, where the metal is found in the cytosol in superoxide dismutase see Copper Proteins with Type Sites) or metallothionein see Metallothiondns) In response to a copper challenge, yeast adaptively synthesizes metallothionein to detoxify the metal. Copper is also bound, transported, and assimilated into tissues by ceruloplasmin. 

Transcuprin is another transport protein for copper in circulating blood (about 7% binding capacity). Apart from being bound to glutathione and metallothionein. 

The most definitive assessment of the metal composition of metalloproteins comes from the application of element-specific detection methods. CE-ICP-MS provides information not only about the type and quantity of individual metals bound to the proteins but also about the isotopes of each element as well [11,12]. Elemental speciation has become increasingly important to the areas of toxicology and environmental chemistry. Such analytical capability also opens up important possibilities for trace element metabolism studies. Figure 1 depicts the separation of rabbit liver metallothionein containing zinc, copper, and cadmium (the predominant metal) using CE-ICP-MS with a high-sensitivity, direct injection nebulizer (DIN) interface. UV detection (200 nm) was used to monitor the efficiency of the CE separation of the protein isoforms (MT-1 and MT-2), whereas ICP-MS detection made it possible to detect and quantify specific zinc, copper (not shown), and cadmium isotopes associated with the individual isoform peaks. 

For -t-2 cations such as zinc(II) and cadmium(ll) each metallothionein molecule contains up to seven metal atoms. X-ray studies indicate that the metal atoms are in approximately tetrahedral sites bound to the cysteine sulfur atoms. The soft mer-cury(II) ion has a higher affinity for sulfur and will displace cadmium from metallothionein. At first the mercury ions occupy tetrahedral sites but as the number increases, the geometries of the metal sites and protein change until about nine Hg(Il) atoms are bound in a linear (S—Hg—S) fashion.92 Up to twelve + 1 cations such as copper(l) and silver(I) can bind per molecule, indicating a coordination number lower than four, probably three (see Problem 12.34),... 

Most cadmium in urine is bound to metallothionein. This protein occurs in the organism as at least four genetic variants. The two major forms, I and II, are ubiquitous in most organs, particularly in the liver and kidney, and also in the brain. Metallothionein isolated from adult or fetal human livers contained mainly zinc and cooper, whereas that from human kidneys contained zinc, copper, and cadmium. 

Copper is absorbed from food in the upper small intestine. The absorption is primarily dependent on the quantity of the copper present in the diet. High intake of zinc diminishes copper absorption by inducing metallothionein formation in the mucosal cells. Metallothioneins, due to their high affinity for copper, bind it preferentially and the bound copper is lost during the sloughing of cells from the villi. Copper accumulation in patients with Wilson s disease can be reduced by giving oral zinc acetate, which decreases absorption (discussed later). Absorbed copper is transported to the portal blood where it is bound to albumin (and probably transcuprein), amino acids, and small peptides. Copper binds to albumin at the N-terminal tripeptide (Asp-Ala-His) site. The recently absorbed copper is taken up by the liver, which plays a central role in copper homeostasis. 

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