Enzyme ceruloplasmin

The multi-copper carrying enzyme ceruloplasmin (CP), found in large amounts in liver and nervous tissues, has been shown to convert NO to RSNOs. The proposed mechanism involves the binding of NO to the CP type I Cu-sites. The NO is then oxidized to NO+ and transferred to RS giving rise to RSNO (Innoue et al., 1999). 

In in vitro studies penicillamine inhibited angiotensin-con-verting enzyme (ACE) and carboxypeptidase (930). Penicillamine interferes with the functions of the copper-containing enzyme ceruloplasmin, and some of the penicillamine- and copper-containing complexes formed in vivo have a superoxide dismutase effect (931). In patients with scleroderma, penicillamine normalized collagen metabolism, by inhibiting beta-galactosidase activity (932). 

The accumulation of iron is dependent on its transport into the cell. Askwith and Kaplan (Chapter 4) discuss iron transport mechanisms in eukaryotic cells, developing models based on studies carried out in the yeast, Saccharomyces cerevisiae. These cells possess both siderophore-dependent and elemental iron transport systems. The latter system relies on cell surface ferrireductases to convert extracellular ferric chelates to ferrous iron, which can be transported through either a high or low affinity iron transport system. Studies on a high affinity ferrous iron transporter (FET3) revealed that the multicopper oxidase will oxidize ferrous to ferric iron, which is then mobilized across the membrane by a ferric transmembrane permease (Ftrlp). This is a highly specific transport system in yeast it only transports iron. In humans, the copper enzyme, ceruloplasmin, is responsible for the radical-free oxidase activity. This plasma protein oxidizes the ferrous iron that is excreted from cells into the transferrin-usable ferric form. 

In animals, copper plays some specific physiological roles, forming part of many proteins, e.g. the enzymes ceruloplasmin, monoamine oxidase, superoxide dismutase (SOD) and cytochrome c oxidase. Copper deficiency is a more common metabolic alteration than copper toxicosis and anemia is the common expression in most of the studied species. Ruminants appear to be particularly susceptible to copper toxicity and are the only animals in which significant, even lethal, effects can occur (Davis and Mertz, 1986). According to Davis and Mertz (1986), the normal range of copper in the blood of healthy animals is 0.5-1.5 (Jig/ml. However, Minoia et al. (1990) report a slightly different range in humans, from 0.535 to 1.94 jLg/ml. In mammalian blood, 50% of the copper is usually contained in the erythrocytes, where it appears to be more or less firmly bound (Davis and Mertz, 1986). 

The determination of the enzyme ceruloplasmin based on spectrophotometric measurements of the initial rate of /)-phenylenediamine (PPD) oxidation is investigated at a constant enzyme concentration of 13.6 mg/1. In the first step, a screening 2 design is used to study the effects of the factors pH value, temperature, and substrate concentration PPD,... 

Figure 4.10 Factor effects in the kinetic-enzymatic oxidation of p-phenylenediamine (PPD) by the enzyme ceruloplasmin.

Copper is also necessary for the efficient utilisation of iron and for the biosynthesis of some physiologically important compounds, such as the enzyme ceruloplasmin, which is produced in Hver cells and acts in the blood plasma as the main Cu compound, but it is not involved in copper transport to the target organs. Plasma copper is transported primarily bound to albumin and partly in the form of complexes with low molecular weight ligands, such as histidine. Ceruloplasmin also has the catalytic activity of ferro oxidase, which means that it catalyses the oxidation of Fe " " ions absorbed in the blood plasma to Fe + ions, thus allowing fixation of iron in the transferrin molecule. Copper deficiency, therefore, is similar to iron deficiency and leads to anaemia. 

Copper is an essential trace element. It is required in the diet because it is the metal cofactor for a variety of enzymes (see Table 50—5). Copper accepts and donates electrons and is involved in reactions involving dismu-tation, hydroxylation, and oxygenation. However, excess copper can cause problems because it can oxidize proteins and hpids, bind to nucleic acids, and enhance the production of free radicals. It is thus important to have mechanisms that will maintain the amount of copper in the body within normal hmits. The body of the normal adult contains about 100 mg of copper, located mostly in bone, liver, kidney, and muscle. The daily intake of copper is about 2—A mg, with about 50% being absorbed in the stomach and upper small intestine and the remainder excreted in the feces. Copper is carried to the liver bound to albumin, taken up by liver cells, and part of it is excreted in the bile. Copper also leaves the liver attached to ceruloplasmin, which is synthesized in that organ. 

Solberg and co-workers have applied discriminate analysis of clinical laboratory tests combined with careful clinical and anatomic diagnoses of liver disease in order to determine which combinations of the many dozen liver diagnostic tests available are the bes t ( ). These authors found that the measurement of GPT, GMT, GOT, ALP and ceruloplasmin were the most useful enzymatic tests, when combined with other non-enzymatic tests such as the measurement of bilirubin, cholesterol, hepatitis-B associated Australian antigen, etc. Another group of highly useful enzymes, not discussed in this review, are those clotting factors and the enzyme cholinesterase which are synthesized by the liver cells. 

Catalytic reduction of oxygen directly to water, while not as yet possible with traditional catalyst technology at neutral pH, is achieved with some biocatalysts, particularly by enzymes with multi-copper active sites such as the laccases, ceruloplasmins, ascorbate oxidase and bilirubin oxidases. The first report on the use of a biocatalyst... 

Adults require 1-2 mg of copper per day, and eliminate excess copper in bile and feces. Most plasma copper is present in ceruloplasmin. In Wilson s disease, the diminished availability of ceruloplasmin interferes with the function of enzymes that rely on ceruloplasmin as a copper donor (e.g. cytochrome oxidase, tyrosinase and superoxide dismutase). In addition, loss of copper-binding capacity in the serum leads to copper deposition in liver, brain and other organs, resulting in tissue damage. The mechanisms of toxicity are not fully understood, but may involve the formation of hydroxyl radicals via the Fenton reaction, which, in turn initiates a cascade of cellular cytotoxic events, including mitochondrial dysfunction, lipid peroxidation, disruption of calcium ion homeostasis, and cell death. 

The copper transport function of ceruloplasmin has been documented in several reviews (e.g. see refs. 15, 42, 43) and a transport function established. The turnover of ceruloplasmin allows copper ions to move from the major sites of ceruloplasmin synthesis in liver cells [44,45] to peripheral tissues for incorporation into copper-dependent enzymes [46,47], but transport mechanisms may also be active which involve copper atoms in the intact protein. However, the complexity of the protein has made it difficult to determine which, if any, of the six integral copper atoms are involved in copper delivery or whether there exist additional... 

The present volume is the fourth in the series and covers the topics lithium in biology, the structure and function of ceruloplasmin, rhenium complexes in nuclear medicine, the anti-HIV activity of macrocyclic polyamines and their metal complexes, platinum anticancer dmgs, and functional model complexes for dinuclear phosphoesterase enzymes. The production of this volume has been overshadowed by a very sad event—the passing away of the senior editor, Professor Robert W. Hay. It was he who conceived the idea of producing this series and who more than anyone else has been responsible for its continuation. A tribute by one of his many friends, Dr. David Richens, is included in this Volume. 

Copper is part of several essential enzymes including tyrosinase (melanin production), dopamine beta-hydroxylase (catecholamine production), copper-zinc superoxide dismutase (free radical detoxification), and cytochrome oxidase and ceruloplasmin (iron conversion) (Aaseth and Norseth 1986). All terrestrial animals contain copper as a constituent of cytochrome c oxidase, monophenol oxidase, plasma monoamine oxidase, and copper protein complexes (Schroeder et al. 1966). Excess copper causes a variety of toxic effects, including altered permeability of cellular membranes. The primary target for free cupric ions in the cellular membranes are thiol groups that reduce cupric (Cu+2) to cuprous (Cu+1) upon simultaneous oxidation to disulfides in the membrane. Cuprous ions are reoxidized to Cu+2 in the presence of molecular oxygen molecular oxygen is thereby converted to the toxic superoxide radical O2, which induces lipoperoxidation (Aaseth and Norseth 1986). 

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

Different chemical environments surrounding the T1 copper result in different redox potentials. Fungal laccases demonstrate the highest potential, close to the equilibrium potential of oxygen reduction in their respective pH regions (see Table 1). Laccases, however, are anion sensitive, with deactivation involving dissociation of T2 copper from the active site of the enzyme. Alternative copper oxidases such as bilirubin oxidase and ceruloplasmin ° ... 

A blue, copper-containing glycoprotein present in mammalian blood plasma and containing type 1, type 2, and type 3 copper centers. The type 2 and type 3 copper centers are close together, forming a trinuclear copper cluster. Ceruloplasmin has an important role in the transport and storage of copper ions. Thus, it participates in the metabolism of copper-containing enzymes. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

Recent publications signal the continued interest in the function of this protein. It has been called a stress enzyme, involved in influenza virus infection (Tomas and Toparceanu, 1986). An explanation for Wilson s disease in terms of a genetic defect resulting in failure to convert from a neonatal (i.e., low) level of ceruloplasmin and copper to a normal adult level has been reported (Srai et al., 1986). Tissue specificity for the binding of ceruloplasmin to membranes was demonstrated in a study investigating the possible role of ceruloplasmin-specific receptors in the transfer of copper from ceruloplasmin to other copper-containing proteins (Orena et al, 1986). Ceruloplasmin has been shown to be effective in transferring copper to Cu,Zn-SOD in culture (Dameron and Harris, 1987), as has copper albumin. In view of the variable content of copper in this protein, it is not clear which copper is transferred. 

The oxidation of dopamine by ceruloplasmin yielded a free radical with a very short lifetime, characterized by an EPR signal at g = 2.006 Dimethyl-p-phenylenediamine was oxidized faster by the enzyme to the free radical Wurster s red, which was further oxidized by the enzyme in another one-electron step. The free radical seemed, moreover, to be stabilized by ceruloplasmin... 

The storage role of (Cu,Zn)-SOD in seeds e.g. seems plausible, when the Cu-carrier function of ceruloplasmin is considered The lipophilic anti-inflammatory and anti-ulcer Cu-chelates could also raise the Cu concentration in certain tissues and thus enhance their lysyl oxidase activity. But especially Cu(acetylsalicylate)2 inhibited protine,2-oxoglutarate dioxygenase (EC 1.14.11.2) and lysine,2-oxoglutarate dioxygenase (EC 1.14.1.4), which are also important enzymes in the processing of collagen... 

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