Spectral and functional properties of ceruloplasmin under UV irradiation

During oxydase reaction spectral characteristics of ceruloplasmin at absorption of copper ions and protein part of the molecule are shown to change. It has been ascertained, that when irradiating ceruloplasmin by UV-light the functioning of intramolecular electron transport chain is broken, the degree of positive cooperativity (a Hill's constant) on substrate decreases. It is supposed, that these changes are caused by disturbance of interdomain interactions in a protein molecule.     

Purification and partial characterization of ceruloplasmin from chicken serum

The preparation and properties of ceruloplasmin from chicken serum are described. Ethanol-CHCl3 was used to precipitate the crude protein, followed by adsorption and elution from DEAE-Sephadex. Further treatment with Sephadex G-200 and CM-Sephadex yielded an intensely blue protein judged 1572-fold purer than starting serum. epsilon-Aminocaproic acid (0.02 M) was present in all buffers and starting sera. Chicken ceruloplasmin appears to be a single polypeptide, apparent Mr 124,000, with an A610/A280 ratio of 0.07 and an absorption maximum at 602 nm. Hexose, hexosamine, and sialic acid accounted for 7.2% of the weight; copper represented 0.20%, which suggested four or five copper atoms per molecule. Chicken ceruloplasmin catalyzed the azide-sensitive oxidation of p-phenylenediamine (PPD) and N,N'-dimethyl-p-phenylenediamine (DPD), and showed ferroxidase activity similar to that of human ceruloplasmin. Its amino acid composition, although similar in many residues to human ceruloplasmin, was decidedly lower in methionine and tyrosine. The chicken protein had one-third the sialic acid content of human ceruloplasmin and showed immunochemical nonidentity with human ceruloplasmin.     

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.     

Horse plasma ceruloplasmin molecular weight and subunit analysis

Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin. There are several reports demonstrating that ceruloplasmin is made up of multiple chains. Ryden has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial preparations of the protein. By introducing epsilon-aminocaproic acid, a general protease inhibitor, at the beginning of the enzyme preparation, Ryden proposed a single-chain structure for ceruloplasmin. On the contrary the results presented by Freeman and Daniel showed that human ceruloplasmin is a multichain protein. In this paper we report a new purification method for horse ceruloplasmin which furnishes a homogeneous protein preparation in high yield and with good reproducibility. This procedure allowed to determine with greater accuracy the molecular mass of the protein, of 120,000 daltons by gel chromatography and 115,000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse ceruloplasmin is a glycoprotein containing about 20% carbohydrate by weight.     

Localization of active sites in human ceruloplasmin from data of intra- and intermolecular homology

The identification of possible copper ligands in human ceruloplasmin was carried out by the computer similarity analysis for sequences of ceruloplasmin and several other copper oxidases: azurin, plastocyanin, superoxide dismutase, tyrosinase and hemocyanin. It follows from the analysis of inter- and intramolecular homology that copper active sites of different types appeared to be in close contacts within the ceruloplasmin molecule.     

Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin

To examine the mechanisms of copper incorporation during ceruloplasmin biosynthesis, we developed methods to resolve and identify apo and holoceruloplasmin. The identity of holoceruloplasmin was confirmed by oxidase activity staining, immunoblotting, 67Cu-ligand exchange, and 67Cu-ligand blotting. Following metabolic labeling of human liver and lung cell lines with 67Cu, newly synthesized holoceruloplasmin was detected in the culture media as two species with apparent molecular masses of 84 and 79 kDa. Pulse-chase studies demonstrate that exogenous copper is readily available for incorporation into newly synthesized ceruloplasmin and that the kinetics of apo and holoceruloplasmin synthesis and secretion are identical. Inhibition of N-linked glycosylation did not affect the rate or amount of copper incorporated into newly synthesized ceruloplasmin but did result in the secretion of a single 68-kDa holoceruloplasmin moiety. Despite differences in the kinetics of copper uptake between cell lines a linear rate of copper incorporation into newly synthesized ceruloplasmin was observed with no evidence of copper exchange following biosynthesis. Under the conditions studied, holoceruloplasmin accounted for less than 5% of the total ceruloplasmin synthesized and secreted by each cell line. The data indicate that copper is incorporated into newly synthesized ceruloplasmin early in the course of biosynthesis by a process independent of N-linked carbohydrate addition. This process of copper incorporation results in an apparent conformational change in the ceruloplasmin molecule which does not affect the secretory rate of the protein.     

Horse Plasma Ceruloplasmin Molecular Weight and Subunit Analysis

Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin (1). There are several reports demonstrating that ceruloplasmin is made up of multiple chains (2-3-4-5-6-7). Ryden (8) has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial preparations of the protein. By introducing aminocaproic acid, a general protease inhibitor, at the beginning of the enzyme preparation, Ryden proposed a single-chain structure for ceruloplasmin. On the contrary the results presented by Freeman and Daniel (9) showed that human ceruloplasmin is a multichain protein.

In this paper we report a new purification method for horse ceruloplasmin which furnishes a homogeneous protein preparation in high yield and with good reproducibility.

This procedure allowed to determine with greater accuracy the molecular mass of the protein, of 120000 dal-tons by gel cromatography and 115000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse cerulopla-smin is a glycoprotein containing about 20% carbohydrate by weight.     

Effect of pH on the oxidase activity of ceruloplasmin

The effect of pH on the kinetic parameters (Kms, Vs) of the reaction of adrenaline and Fe(II) (More's salt) oxidation by ceruloplasmin isolated from human donor blood was investigated. It was assumed that the imidazole group of histidine is functionally important for the above reactions. For Fe(II) the effect of the ionizeable group was observed during substrate binding to the ceruloplasmin molecule, whereas in the course of the adrenaline oxidation reaction it manifests itself during catalytic interaction of the substrate with the enzyme. The organic substrate can bind both to the protonated and to the non-protonated form of the enzyme. Fe(II) interacts only with the protonated form of the protein. In both cases, the rate-limiting step of the oxidase reaction is preceded by a single step, i.e., proton binding. The schemes describing the order of proton attachment in the course of the above reactions are proposed.     

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers

Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Differences in the expression of the human interferon-gamma gene in fresh lymphocytes and cultured lymphoblasts

Mammalian erythrocytes have been shown to bind 125I labeled ceruloplasmin. Binding was reversible and specific. Scatchard analysis yielded linear plots with a Kd of approximately 5nM. The binding site appeared to be a protein located on the cell surface. A ceruloplasmin binding protein with a molecular weight of 60,000 daltons was isolated from human erythrocytes. Erythrocytes which were not protected by ceruloplasmin's antioxidant properties, did not bind ceruloplasmin. Our results provide evidence for the presence of ceruloplasmin receptors in the erythrocyte membrane. It is proposed that the antioxidant activity of ceruloplasmin may play a role in determining the lifespan of circulating red cells.     

Immunochemical investigation on human ceruloplasmin. Partial explanation of the "heterogeneity".

Human ceruloplasmin from fresh serum has been purified by chromatography on hydroxyapatite and Con A-Sepharose. Quantitative immunoelectrophoretic analysis of fresh serum, stored serum and fractions from the different purification steps for human ceruloplasmin has been carried out. A combination of the latter, advanced technique with amino acid analysis, molecular weight determination by size chromatography, urea treatment, staining for oxidase activity and enzymatic proteolysis, has revealed that: 1) human cerulplasmin is a heterogeneous mixture of two glycoproteins (x) differing only in their carbohydrate content and 2) the protein part contains at least one very labile peptide bond which upon enzymatic hydrolysis gives rise to peptides with molecular weights of 93,000 (y) and 24,000 (z) dalton, respectively. The two glycoproteins are immunochemically identical. The y peptide is immunochemically partially identical, and the z peptide immunochemically non-identical, with the parent molecule. The y and z peptides are non-identical. On the basis of these observations a simplified two-dimensional model of human ceruloplasmin is proposed.     

Ceruloplasmin, an Indicator of Copper Status

For clinical purposes, the non-ceruloplasmin copper fraction is routinely derived on the basis that ceruloplasmin binds six Cu atoms. However, this approach is limited because the actual ceruloplasmin copper binding is unclear. We performed direct measurement of the total serum copper and ceruloplasmin in 790 healthy individuals. We used an immunoprecipitation technique to separate ceruloplasmin and determined Cu content. With these values, we calculated the Cu/ceruloplasmin (Cp) ratio and thus generated data to support or discard the theoretical calculation of the non-ceruloplasmin fraction. Average of serum Cu and Cp levels were 18.4 +/- 4.4 micromol/l and 390 +/- 100 mg/l, respectively. The immunoprecipitation procedure allowed us to calculate a Cu/Cp ratio of 5.8, respectively, which supports the methodology of calculation that assigns a mean of six copper atoms to each ceruloplasmin molecule. With these values, we calculated that, in apparently normal adults, the non-ceruloplasmin copper (NCPC) fraction is lower than 1.3 micromol/l of Cu. In this report, we examine the Cp/Cu ratio by using Cp immunoprecipitation procedure. Our in vitro and in vivo studies indicate that, as a mean, there are 5.8 atoms of Cu per Cp molecule and that <1.3 micromol/l of Cu would correspond to the NCPC.     

Molecular and pathological basis of aceruloplasminemia

Aceruloplasminemia is an autosomal recessive neurodegenerative disease characterized by iron accumulation in the brain as well as visceral organs. It is a loss-of-function disorder caused by mutations in the ceruloplasmin gene. Clinically, this disease consists of the triad of adult-onset neurological disease, retinal degeneration and diabetes mellitus. Massive iron accumulation and extensive loss of neurons are observed in the basal ganglia. The elevated iron concentration is associated with increased lipid peroxidation in the brains of aceruloplasminemia patients. Enlarged or deformed astrocytes and spheroid-like globular structures are characteristic neuropathological findings in aceruloplasminemia. Moreover, deformed astrocytes and globular structures react positively to anti-4-hydroxynonenal antibody, suggesting that increased oxidative stress is involved in neuronal cell death in aceruloplasminemia brain. More than 30 aceruloplasminemia-causing mutations in the ceruloplasmin gene have been identified. We examined the biosynthesis of two missense ceruloplasmin proteins that result from a Japanese P177R mutation and a Dutch G631R mutation, using Chinese hamster ovary cell expression system. The P177R mutant protein is retained in the endoplasmic reticulum. The G631R mutant protein, predicted to alter the interactions at a single type I copper-binding site, prevented incorporation of copper into apoceruloplasmin and resulted in the synthesis and secretion only of apoceruloplasmin. Molecular analysis of missense mutations showed different structure-function relationships in ceruloplasmin protein. The investigation of mutant ceruloplasmin reveals new insights into molecular pathogenesis of aceruloplasminemia as well as biosynthesis, trafficking, and function of ceruloplasmin.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

Alterations of Antioxidant and Oxygen Transport Properties of Blood in Adenocarcinoma-Bearing Mice during Chemotherapy

The influence of murine intestinal adenocarcinoma on the antioxidant status and hemoglobin content in the blood was analyzed. Murine adenocarcinoma was accompanied with a decrease of the nonprotein thiol concentration and an increase in ceruloplasmin. A conformational change in the globin molecule but not in the heme group was revealed in the hemoglobin molecule. The changes in the protein appear to be caused by nonspecific damage that results from the processes of peroxidation.     

Various properties of the ceruloplasmin receptor, isolated from human erythrocyte membranes

An electrophoretically pure preparation of ceruloplasmin (CP) receptor which retains its ability to bind to CP was isolated from human erythrocyte membranes. It was found that in terms of molecular mass, number and size of spontaneously proteolytic fragments as well as antigenicity, the CP receptor molecule strongly resembles that of CP. A comparative analysis of two-dimensional peptide maps of full tryptic digests of the both protein revealed that about 30% of CP peptides are identical in respect of their electrophoretic and chromatographic mobilities which points to the genetic independence of these proteins. The roles of CP and CP receptor in copper metabolism are discussed.     

Isolation and characterization of copper-binding sites of human ceruloplasmin

A tryptic cleavage procedure was used to obtain stable copper-containing peptide regions of human ceruloplasmin. Tryptic digestion-derived copper peptides were fractionated by gel filtration chromatography, yielding two fractions, one with an apparent molecular weight of 11000 and the other 1000. The high molecular weight fraction (11K fraction) was found to contain 50% of the copper atoms of the ceruloplasmin molecule and behaved as a single copper-containing component by gel filtration chromatography and by isoelectric focusing. The low molecular weight fraction (1K fraction) was found to be a mixture of three or four copper peptides by isoelectric focusing. Acrylamide gel electrophoresis studies, amino acid composition analysis and terminal amino acid determinations showed the 11K fraction to be a complex composed of at least three peptides arising from different regions of the ceruloplasmin molecule. Two of the peptides of the 11K complex appear to be derived from the 19K fragment of the ceruloplasmin molecule (18); one peptide in the complex appears to correspond to the aspartic acid-rich portion, residues 7-30, and the other to the histidine-rich portion, residues 103-157. Most preparations of ceruloplasmin are reported to consist of three non-covalently linked fragments that have molecular weights of 67K, 50K and 19K. Dwulet and Putnam (20) proposed a model for the sequence structure of ceruloplasmin where the molecule exhibits a three-fold repeat pattern of two alternating structures, here termed X and Y.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.