Alaskan malamute chondrodysplasia--VII. Isolation and characterization of copper, zinc and iron binding proteins in Canis familiaris.

1. Three low molecular weight (12,000, 10,000 and 7,000) metal binding proteins have been isolated from the livers of normal and chondrodysplastic Alaskan Malamutes. 2. Comparison studies between kennel (mixed breed) dogs and both adult and immature Alaskan Malamutes suggested that the disturbance in trace mineral metabolism found in the Malamutes is almost entirely reflected in the 12,000 mol. wt species. 3. The major copper-inducible protein (10,000 mol. wt) observed in kennel dogs was not found to be inducible in Malamutes and contained constant ratios of both copper and zinc to protein in metal binding proteins isolated from the livers of both normal and dwarf Malamutes. 4. The copper and zinc found in the UM2 concentrates (mol. wt greater than 2000) of immature Malamutes showed very little affinity to the proteins and these metals were found chiefly in a peptide fraction which apparently serves as a reservoir from which the storage proteins obtain the metals that they bind. 5. Regression analysis indicated a statistically significant correlation between both copper and zinc concentrations and the carbohydrate concentration in the proteins investigated.     

"Pulling the plug" on cellular copper: the role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

Metal-binding proteins in bream (Abramis brama L.) caught in the River Elbe

Metal-binding proteins were investigated in livers of bream caught in the River Elbe from Steti (Czech Republic) to Hamburg (Federal Republic of Germany). A major zinc and copper binding protein fraction with a low molecular weight of 10 000 to 12 000 Da and with properties similar to mammalian hepatic metallothionein was isolated from bream livers using gel filtration chromatography. Two protein isoforms could be separated by reversed phase-high performance liquid chromatography (RP-HPLC), however, mercury was associated with only one isoform. The possibility of different detoxification potentials of the isoforms is discussed. Maximal concentrations of metal-binding protein were detected in samples from Dresden. If metal-binding proteins are to be included in a biological monitoring study, further investigations are required.     

Metal-binding proteins in bream (Abramis brama L.) caught in the River Elbe

Metal-binding proteins were investigated in livers of bream caught in the River Elbe from Steti (Czech Republic) to Hamburg (Federal Republic of Germany). A major zinc and copper binding protein fraction with a low molecular weight of 10 000 to 12 000 Da and with properties similar to mammalian hepatic metallothionein was isolated from bream livers using gel filtration chromatography. Two protein isoforms could be separated by reversed phase-high performance liquid chromatography (RP-HPLC), however, mercury was associated with only one isoform. The possibility of different detoxification potentials of the isoforms is discussed. Maximal concentrations of metal-binding protein were detected in samples from Dresden. If metal-binding proteins are to be included in a biological monitoring study, further investigations are required.     

“Pulling the plug” on cellular copper: The role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

Analysis of hepatic copper, zinc, metallothionein and metallothionein-Ia mRNA in developing sheep

The concentrations of zinc, copper, metallothionein and metallothionein-Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30-40 days gestation) had very high concentrations of hepatic zinc (2305 +/- 814 micrograms/g dry mass), and that these levels declined steadily to 644 +/- 304 micrograms/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein-Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261 +/- 94 molecules/pg RNA to 71 +/- 18 molecules/pg near to term. Metallothionein-Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 micrograms/g (wet mass) in one foetus on day 34 of gestation to 862 micrograms/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 micrograms/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein-Ia mRNA decreased from 1.3 to 3.2 x 10(5) molecules metallothionein/molecule of metallothionein-Ia mRNA during gestation to between 0.28-0.64 x 10(5) molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.     

Similarities of physico-chimical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Similarities of physico-chemical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested the the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Hepatic metallothioneins in two neotenic salamanders,Proteus anguinus and Necturus maculosus (Amphibia,Caudata)

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.     

Activation of protein-bound copper ions during early glycation: study on two proteins

This study proposes several possible pathways by which hyperglycemia could make protein-bound metal ions more redox active. These mechanisms were tested on bovine serum albumin and calf lens protein. Proteins rich in early glycation products were less capable of competing for copper ions in the presence of other ligands (e.g., glycine and calcein), suggesting that glycated proteins might have diminished stability constants of their copper chelates compared to control counterparts. When protein-copper complexes were tested for their ability to cause the oxidation of ascorbic acid, as well as the reduction of molecular oxygen to hydrogen peroxide, glycated and control proteins differed considerably in their redox abilities. Oxidative damage on proteins documented by protein carbonyl content and amino acid analysis indicates the involvement of Fenton chemistry upon metal chelation. The possible biological consequences of the observed activation of metal ions bound to early glycated proteins are discussed.     

Bioinformatic identification of the most ancient copper protein architecture

Since copper ions participate in many cellular processes and are implicated in pathogenesis of many diseases, copper proteins have important biological significance. Thus, it is of interest to explore their origins, especially to address the following question: which is the most ancient architecture of copper proteins? In this paper, through analyzing the architectural features of copper proteins, we find that the fold-domain relationship of these proteins follows a power law, which can be explained by preferential attachment principle and implicates that the architecture of the most ancient copper proteins belonged to Cupredoxin-like (b.6) fold. According to the chronology of protein folds, this architecture originated rather late, which can be understood in terms of the low abundance of reducing amino acids (e.g., His, Cys and/or Met) in the primordial world, because these amino acids are required by copper proteins to bind copper ions.     

Isolation and characterization of a blue copper protein from Thiobacillus versutus

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1.     

The effect of copper deficiency on the formation of hemosiderin in sprague-dawley rats

We demonstrated previously that loading iron into ferritin via its own ferroxidase activity resulted in damage to the ferritin while ferritin loaded by ceruloplasmin, a copper-containing ferroxidase, was not damaged and had similar characteristics to native ferritin (Welch et al. (2001) Free Radic Biol Med 31:999–1006). Interestingly, it has been suggested that the formation of hemosiderin, a proposed degradation product of ferritin, is increased in animals deficient in copper. In this study, groups of rats were fed normal diets, copper deficient diets, iron supplemented diets, or copper deficient-iron supplemented diets for 60 days. Rats fed copper-deficient diets had no detectable active serum ceruloplasmin, which indicates that they were functionally copper deficient. There was a significant increase in the amount of iron in isolated hemosiderin fractions from the livers of copper-deficient rats, even more than that found in rats fed only an iron-supplemented diet. Histological analysis showed that copper-deficient rats had iron deposits (which are indicative of hemosiderin) in their hepatocytes and Kupffer cells, whereas rats fed diets sufficient in copper only had iron deposits in their Kupffer cells. Histologic evidence of iron deposition was more pronounced in rats fed diets that were deficient in copper. Additionally, sucrose density-gradient sedimentation profiles of ferritin loaded with iron in vitro via its own ferroxidase activity was found to have similarities to that of the sedimentation profile of the hemosiderin fraction from rat livers. The implications of these data for the possible mechanism of hemosiderin formation are discussed.     

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.     

Small copper-binding proteins from normal and copper-loaded liver.

The same three low molecular weight copper-binding proteins which have been purified from the soluble fractions of Cu2+-loaded livers (200 mug Cu2+ per g wet weight) were also found to be present in livers of normal rats (4 mug Cu2+ per g wet weight) but in much smaller quantities. Therefore, Cu2+ loading enhances the amount of preexisting proteins rather than inducing the synthesis of new proteins. Amino acid analyses of each of the three showed them to be different from the metallothioneins which are present on loading with other trace metals including Cd2+ and Zn2+.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Blue copper proteins: a comparative analysis of their molecular interaction properties

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.