Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.     

Magnetic susceptibility of laccases and ceruloplasmin.

1. Recent magnetic susceptibility measurements on laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera showed a deviation from Curie behaviour above 50 K, which was taken as evidence for an antiferromagnetically coupled Cu(II)-Cu(II) pair in the oxidized enzyme. The magnetic susceptibility of this protein has been reinvestigated. Further measurements on laccase from the fungus Polyporus versicolor and human ceruloplasmin (iron(II):oxygen oxidoreductase, EC 1.16.3.1) are presented. 2. The magnetic susceptibility of fungal laccase and lacquer tree laccase can be accounted for by the EPR detectable copper ions in the temperature range 40--300 K. 3. If an antiferromagnetically coupled Cu(II)-Cu(II) pair exists in the laccases, then the coupling, expressed as --J, should be at least of the order of 300 cm-1, as deduced from the Curie dependence of the susceptibility and the sensitivity in our measurements. 4. If an analogy with the laccases is assumed for the EPR invisible copper in ceruloplasmin then a limiting value of the coupling may be deduced also in this case, with --J at least of the order of 200 cm-1.     

Cloning and expression analysis of the sheep ceruloplasmin cDNA.

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.     

Characteristics of ceruloplasmin synthesis in mammalian embryogenesis. 2. The yolk sac as the site of the primary expression of the ceruloplasmin gene in rats

It was shown that the omphaloid placenta and, first of all, visceral wall of yolk sac is the site of primary synthesis of ceruloplasmin (CP), whereas the activation of CP synthesis in the liver cells is secondary and is revealed from the 12th day of embryo-genesis. The CP synthesis in the yolk sac cells proved by selective CP localization in the cells of the yolk sac visceral wall and, first of all, in the cells of visceral endoderm on sections stained by the method of indirect immunofluorescence and using the reaction of soluble peroxidase-antiperoxidase complex. A specific CP-mRNA has been revealed in the yolk sac cells which is actively translated in the polyribosomes isolated from the yolk sac and in the cell-free translation system from the rabbit reticulocytes. on the 14th day of embryogenesis CP amounts to ca. 4% of all polypeptides secreted by the yolk sac cells. As the embryogenesis proceeds, the relative rate of CP synthesis progressively decreases in the yolk sac and increases in the liver cells. CP synthesized by the yolk sac cells has a molecular mass of ca. 122 kD. Possible causes of differences between the "embryonic" and "adult" rat CPs are discussed. A suggestion has been put forward that the time of activation of CP synthesis coincides with the yolk sac formation (8-9th days of embryogenesis) and the cells of visceral endoderm are the site of primary expression of the CP gene.     

Electron microscope study on human ceruloplasmin.

Electron microscopy of human ceruloplasmin (CP) molecules revealed a few distinctive types of particle images. Analysis of these images allows to propose a tentative model for CP: six "subunits" (which we call domains) not much different in size are arranged with 32 point group pseudosymmetry. The determination of the number of polypeptides arising at the spontaneous specific proteolytic fragmentation of CP and their molecular weights conform with this assumption. The electrophoretic studies of the CP samples prepared both with and without potent proteolytic inhibitor, PMSF, revealed that CP is a single-chain protein with molecular weight of 130 000. Isolated and stored without PMSF the polypeptide chain of CP undergoes specific proteolytic cleavage which results in the appearance of polypeptides with molecular weights of 16 000, 48 000, and 64 000. The latter two polypeptides degradate to about two- and three-fold decreased molecular weights fragments, respectively. Therefore, the single polypeptide chain of CP contains at least five peptide bonds which are particularly susceptible to proteolytic attack and which connect six principal segments of the chain. The hydrolysis of these bonds results in liberation of the six fragments which were integrated in the enzymatically active globule of CP.     

Copper regulation of ceruloplasmin in copper-deficient rats.

In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.     

Inhibition of endothelial nitric-oxide synthase by ceruloplasmin.

The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction.     

Reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate

The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five paramagnetic copper ions, two of which, X and Y, not involved in enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the two type-I copper atoms when added in large excess to the enzyme or the anion treated enzyme.     

Oxidative modification of human ceruloplasmin by peroxyl radicals.

Ceruloplasmin (CP), the blue oxidase present in all vertebrates, is the major copper-containing protein of plasma. We investigated oxidative modification of human CP by peroxyl radicals generated in a solution containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When CP was incubated with AAPH, the aggregation of proteins was increased in a time- and dose-dependent manner. Incubation of CP with AAPH resulted in a loss of ferroxidase activity. Superoxide dismutase and catalase did not protect the aggregation of CP, whereas hydroxyl radical scavengers such as ethanol and mannitol protected the protein aggregation. The aggregation of proteins was significantly inhibited by the copper chelators, diethyldithiocarbamate and penicillamine. Exposure of CP to AAPH led to the release of copper ions from the enzyme and the generation of protein carbonyl derivatives. Subsequently, when the amino acid composition of CP reacted with AAPH was analyzed, cysteine, tryptophan, methionine, histidine, tyrosine, and lysine residues were particularly sensitive.     

Uptake of 67Cu by ceruloplasmin in vitro.

Solutions of human ceruloplasmin, in Tris buffer at pH values between 6 and 7.5, readily took up carrier-free 67Cu2+, but only when the substrate, rho-phenylenediamine, was added.