Cytochrome c oxidase, Cu,Zn-superoxide dismutase, and ceruloplasmin activities in copper-deficient bovines

The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 μmol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.     

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identifiedin the liver of rats made copper-deficient by dietary restriction. In homogenates ofsuch livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoreticprofile with respect to the native enzyme. When rat liver extracts were treated withexogenous copper an electrophoretic pattern resembling the native one was observed.Enzyme purified by chromatography on DE-52 resin shows two major components, onecorresponding to genuine, native enzyme and another one, eluting at higher ionicstrength. The latter protein (Fraction II) consists of several isoforms which showthe same characteristics of the native superoxide dismutase as far as immunoreactivityand molecular weight are concerned, but with decreased contents of copper and zinc. Itscatalytic constant, referring to copper content, was 15 times lower than that obtainedfor the native enzyme. Moreover, the catalytic power of purified Fraction II was notregained upon incubation with copper. The occurrence of a superoxide dismutase voidof metals confirms the hypothesis that this protein plays a dual physiological role:in metal metabolism and in superoxide anion dismutation.     

Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells

The objective of this research was to develop a method for measuring Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (E.C. 1.15.1.1) in HL-60 cells and subsequently examine the relationship between cellular copper levels and the activity of this copper-requiring enzyme. In cells such as the neutrophil or HL-60 promyelocyte cell line, the activity of Cu/Zn-SOD cannot be measured because of an increase in the oxidation rate of the substrate by some unknown compound in the cells. Others have utilized heat treatment to inactivate the responsible compounds, however, we found that heat treatment of HL-60 cells resulted in a loss of over half of the activity of the enzyme. The method described here utilizes sodium azide to inhibit the substance(s) that are responsible for the enhanced rate of pyrogallol's oxidation. Gel filtration data confirmed that the compound responsible for the enhanced rate of pyrogallol oxidation was sensitive to azide and did not affect Cu/Zn-SOD activity. When HL-60 cells were incubated with various levels of copper, Cu/Zn-SOD activity did not reflect the cellular copper levels.     

Effect of nutritional copper deficiency on carrageenin edema in the rat

The effects of nutritional copper deficiency on carrageenin edema in the rat were investigated with emphasis on studying the correlation between the degree of copper deficiency and the degree of edema. Carrageenin paw edema in both copper-sufficient and copper-deficient groups of rats was compared after either 20, 40, or 60 d on respective diets. The degree of copper deficiency was quantitated by analyzing total copper concentrations in a number of tissues. Other copper dependent parameters were also determined. Results indicated that: (1) although copper sufficient rats showed relatively little change in the degree of edema, copper-deficient rats showed a steady and significant increase in edema from d 20 to 40 to 60; (2) paw edema in copper-deficient animals was highly and negatively correlated to the concentrations of copper in the liver; the correlation with liver Cu,Zn-superoxide dismutase activity, however, was inconsistent; (3) paw edema was not correlated either to copper concentration in tissues other than liver or to plasma ceruloplasmin activity; and (4) aggravation of carrageenin edema in copper-deficient animals seemed to be mediated via an as yet unknown secondary effect of copper deficiency.     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

Oxygen-independent induction of enzyme activities related to oxygen metabolism in yeast by copper

Aerobic growth of Saccharomyces cerevisiae in the presence of CuSO4 (between 0.1 and 1 mM) caused a generalized induction of major enzyme activities involved in 'housekeeping' routes of oxygen metabolism (cytochrome oxidase, glutathione peroxidases and catalase) which were comparable to or higher than that observed with Cu,Zn-superoxide dismutase. Fumarase and glutathione transferase, tested as controls for oxygen-unrelated activities, were found to decrease under the same conditions. In the absence of oxygen, copper addition to yeast resulted in significant increases of Cu,Zn-superoxide dismutase and glutathione peroxidases and a slight increase of cytochrome oxidase, with catalase remaining undetectable irrespective of whether or not copper was present. Other metal ions tested (Mn2+, Co2+) were unable to produce such effects. It is concluded that copper has a general inducing effect on enzymes related to metabolism of oxygen and oxygen derivatives, which is mediated neither by formation of O2-. and H2O2 nor by interaction with copper-specific apoproteins. These results point to a general role of copper as regulator of the expression of major enzyme activities involved in biological oxygen activation.     

Mitochondrial membrane potential is reduced in copper-deficient C2C12 cells in the absence of apoptosis

Mitochondrial membrane potential is reduced in copper-deficient rat hearts, but it is uncertain if this will lead to the onset of apoptosis. To determine if copper deficiency per se leads to apoptosis, C2C12 cells were made copper deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA-treated cells, the activity of Cu, Zn-superoxide dismutase and cytochrome-c oxidase decreased dramatically. The protein levels of nuclear-encoded subunits of the cytochromie-c oxidase decreased, but the mitochondrial-encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper deficiency.     

Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome

Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce.     

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.     

Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

Neointima formation in the rat carotid artery is exacerbated by dietary copper deficiency

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 microg Cu/g) or deficient (CuD; 0.3 microg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 +/- 0.93 vs 1.45 +/- 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.     

Comparative antioxidant enzyme study in freshwater fishes. I. Distribution of superoxide dismutase, peroxide-decomposing enzymes and lipid peroxidation in herbivorous fishes

The activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase were comparatively studied in organ homogenates from two herbivorous fishes, viz., the grass carp and the silver carp. 2. The protein contents and lipid peroxidation of organ homogenates were also compared. The comparative measurements primarily provide control values for subsequent toxicological examinations. 3. The highest total superoxide dismutase activities were found in the kidney, spleen and liver in the grass carp, and in the kidney and liver in the silver carp. 4. The antioxidant enzyme activities and other parameters of the organ homogenates appear to be independent of the feeding mode, but are rather characteristic of the fish variety.     

Superoxide dismutase, glutathione peroxidase and catalase in developing rat brain.

The specific activities of Cu,Zn- and Mn-superoxide dismutases, of glutathione peroxidase and of catalase, the enzymes considered to be specifically involved in the defence of the cell against the partially reduced forms of oxygen, were determined as the function of postnatal age in the early (up to 60 days) period of rat brain development. The enzymes were assayed in the cytoplasmic fraction, in the crude mitochondrial fraction including peroxisomes, and in the mitochondria. The results show that the temporal changes of these enzymes cannot be correlated with each other, thus indicating that they do not concertedly parallel the increasing activity of aerobic brain metabolism during development. Specifically the cytoplasmic fraction shows a gradual increase of the Cu,Zn-superoxide dismutase activity with age, whereas the glutathione peroxidase activity is constant from birth. Furthermore the increase of the mitochondrial Mn-superoxide dismutase as a function of postnatal age is more remarkable than that of the cytoplasmic Cu,Zn-enzyme. Higher activities of catalase in adult animals are detectable only in the subcellular fraction containing peroxisomes, because of the modest catalase activity of the brain. These results indicate independent regulation of the expression of these enzyme activities in the process of brain differentiation and point to a relative deficiency of enzymic protection of the brain differentiation and point to a relative deficiency of enzymic protection of the brain against potentially toxic oxygen derivatives. This situation is similar to the pattern already described in the rat heart and in rat and mouse ascites-tumour cells, at variance with the much more efficient enzyme pattern present in rat hepatocytes.     

Variable response of selected cuproproteins in rat choroid plexus and cerebellum following perinatal copper deficiency

Recent immunohistochemical characterization of the copper transport protein, Ctr1, reported enriched levels in mouse choroid plexus, and enhancement by copper deficiency. To extend and confirm this, experiments were conducted with Holtzman rats. Following perinatal copper deficiency there was an 80% reduction in brain copper of 24-27 day old copper-deficient (Cu-) rat pups compared to copper-adequate (Cu+) controls. Choroid plexus immunoblot analysis with rabbit anti-hCtr1 demonstrated a 50% higher Ctr1 protein expression in Cu-samples. However, levels of copper chaperone for superoxide dismutase (CCS) were unchanged, suggesting that Ctr1 buffers the choroid plexus against copper deficiency, since CCS normally is much higher in Cu-tissues. There were 13% lower levels of cytochrome c oxidase subunit IV (COX IV) detected in Cuchoroid plexus. In contrast, in cerebellum of Cu-rats CCS was 2-fold higher and COXIV 1.7-fold lower than Cu+ rats consistent with severe copper deficiency. Brain mitochondria from Cu-rats had severe reductions in COXIV content and CCO activity and modest but significant elevations in CCS and reductions in Cu, Zn-superoxide dismutase. COXIV may be a more sensitive marker for copper deficiency than CCS and may prove useful to assess copper status.     

Copper chaperones,intracellular copper trafficking proteins. Function,structure, and mechanism of action

This review summarizes findings on a new family of small cytoplasmic proteins called copper chaperones. The copper chaperones bind and deliver copper ions to intracellular compartments and insert the copper into the active sites of specific partners, copper-dependent enzymes. Three types of copper chaperones have been found in eukaryotes. Their three-dimensional structures have been determined, intracellular target proteins identified, and mechanisms of action have been revealed. The Atx1 copper chaperone binds Cu(I) and interacts directly with the copper-binding domains of a P-type ATPase copper transporter, its physiological partner. The copper chaperone CCS delivers Cu(I) to Cu,Zn-superoxide dismutase 1. Cox17 and Cox11 proteins serve as copper chaperones for cytochrome c oxidase, a copper-dependent enzyme.     

Nutritional copper deficiency does not affect sponge granuloma formation in the rat

Sponge granuloma formation was compared in copper-deficient and copper-sufficient rats following feeding of respective diets for 20, 40, or 60 d. Body weight, total blood hemoglobin, and activities of ceruloplasmin and Cu, Zn-superoxide dismutase in plasma were monitored to ascertain copper deficiency. Mean granuloma weights (mg +/- SEM) in copper-deficient and copper-sufficient groups of rats, respectively, were as follows: 37 +/- 2 and 38 +/- 2 after 20 d, 22 +/- 2 and 23 +/- 2 after 40 d, and 19 +/- 1 and 21 +/- 1 after 60 d on respective diets. Thus, nutritional copper deficiency did not have an effect on sponge granuloma formation in the rat.     

Superoxide dismutase activity in nerve cell culture

Superoxide dismutase (EC 1.15.1.1) activity was assayed in preparations obtained from several clonal lines of nerve cell culture, by enzymatic and nonenzymatic assays. The enzyme was found in dialyzed homogenates of washed cells and in partially purified fractions. The enzyme was also found in cells which had been grown in media containing 5-bromodeoxyuridine, where cell differentiation was observed.     

The Antiozonant Ethylenediurea Does Not Act via Superoxide Dismutase Induction in Bean

It has been proposed that the mode of action of ethylenediurea, a very effective antiozonant, is via an increase in the antioxidant enzyme superoxide dismutase (EH Lee, JH Bennett [1982] Plant Physiol 69: 1444-1449). Data presented here refute that hypothesis. No ethylenediurea-associated increases in Cu/Zn-superoxide dismutase or Mn-superoxide dismutase activity, nor in steady-state Cu/Zn-superoxide dismutase protein levels, were found in soluble extracts of bean (Phaseolus vulgaris L. cv Bush Blue Lake 290) leaves. However, the cytosolic Cu/Zn-superoxide dismutase increased as a result of ozone fumigation and subsequent injury. Also noted was a developmentally related difference between chloroplastic and cytosolic Cu/Zn-superoxide dismutase, the latter declining during maturation of the leaf.