Human Ceruloplasmin Research Articles

Purification and partial characterization of ceruloplasmin from chicken serum

The preparation and properties of ceruloplasmin from chicken serum are described. Ethanol-CHCl 3 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. ϵ-Aminocaproic acid (0.02 m) was present in all buffers and starting sera. Chicken ceruloplasmin appears to be a single polypeptide, apparent M r 124,000, with an A 610 A 280 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.

Interaction of phenothiazine drugs with human ceruloplasmin: Relation between activation of catecholamine neurotransmitter oxidation and electron donating ability of phenothiazine drugs

The influence has been studied of 11 phenothiazine drugs on the oxidation of the catecholamine neurotransmitters noradrenaline and dopamine, catalyzed by human ceruloplasmin. The phenothiazine drugs were not transformed by the enzyme. This makes participation of free phenothiazine radical cations in the oxidation of the catecholamines unlikely. A relation between the electron donating capacity of the phenothiazine drugs and the activating effect on the enzyme has been observed. It is proposed that in the interaction of phenothiazine drugs with ceruloplasmin, charge transfer complexes between the phenothiazines and Cu 2+ of the enzyme, are involved. The possible relevance of charge transfer complexes as a model for the receptor interactions of phenothiazine drugs is discussed.

Automated tandem high-performance liquid chromatographic system for separation of extremely complex peptide mixtures

We have developed an automated tandem chromatography system, which consists of a combination of ion-exchange column chromatography and reversed-phase column chromatography. The system is composed of two independent high-performance liquid chromatography assemblies, in each of which programmed elution is carried out by a computer-assisted controller. A peptide mixture is applied to an ion-exchange column and is eluted in a stepwise manner. The eluent from the first column is introduced directly into the second, reversed-phase column, which is connected in tandem through a tee tube. After application of two column volumes of the eluent, reversed-phase chromatography is performed by linear gradient elution. Stepwise elution for ion-exchange chromatography and the gradient elution for reversed-phase chromatography are synchronized by a computer program. The resolving power and the reproducibility of the method were tested by using a tryptic digest of human ceruloplasmin [molecular weight 132 000 daltons (132 kDa)]. By this method, the digest was resolved reproducibly into several hundred peaks within 16 h. All of the four glycopeptides expected to be obtained by tryptic digestion were purified easily from the whole digest of the protein. Comparison of the peptide maps between a single-chain and a degraded form of ceruloplasmin facilitated the identification of two tryptic peptides, derived from the carboxyl-terminal regions of 67 kDa and 50 kDa fragments of the degraded form, which lack the carboxyl-terminal arginine and lysine residues, respectively. The method may be applicable to comparative peptide mapping of very large proteins exhibiting molecular microheterogeneity, such as carbohydrate or genetic variants; it also can be used complementarily for sequence support of DNA sequencing as well as for preparative purification of peptides as a strategy of protein sequencing of very large proteins.

Secretion of ceruloplasmin by a human clear cell carcinoma maintained in nude mice

Ceruloplasmin is the best known but least understood copper protein. Studies preliminary to investigating the control of ceruloplasmin synthesis have utilized a human renal cell carcinoma maintained in nude mice for 73 passages over a 5-year period. In vitro cultures of these cells were accomplished and the mRNAs were extracted prior to microinjection into Xenopus oocytes. The media examined by SE-HPLC and immunological techniques demonstrated that (1) after in vitro culture, ceruloplasmin was secreted as an uncleaved polypeptide chain with a MW of 135,000; (2) the translational product of ceruloplasmin mRNA injected into Xenopus oocytes was cleaved into fragments with MWs of 110,000, 67,000, and 50,000. The results indicate that mRNA for human ceruloplasmin can be obtained to serve as a template for the synthesis of a cDNA probe to investigate the control of human ceruloplasmin's synthesis.

Origins of biliary copper.

We tested the hypothesis that the copper present in bile--the major route of elimination of the metal from the body--is derived exclusively from hepatocytes by administering radiocopper (64Cu or 67Cu)-labeled ionic Cu, desialylated (AsCPN) or intact human ceruloplasmin (CPN), intravenously, to rats with cannulated bile ducts. The rates of appearance and the total amounts of radiolabeled isotope recovered in bile were measured. The three vehicles chosen for the delivery of radiocopper interact differently with hepatocytes: ionic Cu is taken up by a passive process (Schmitt, R. C. et al., Am. J. Physiol. 1983; 244:G183-G191); AsCPN is promptly cleared from the circulation by specific receptor-mediated endocytosis, and CPN largely remains in the circulation for the duration of the experiment. Similar amounts of radiocopper were recovered following injections of ionic Cu (3.1%) or CPN (2%), but substantially larger amounts (8.1%) were excreted after administration of AsCPN. Using an antibody to CPN which reacts also with AsCPN, we found about 70% of the bile radioactivity to be immunoprecipitable following injections of either glycoprotein, indicating that a fraction of these copper proteins had entered the bile essentially unmodified. Our observations indicate that in addition to the lysosomal compartment which catabolizes a portion of the AsCPN in hepatocytes, there appears to be a direct route for AsCPN from hepatocellular sinusoids to the bile canaliculi. Since CPN does not interact significantly with hepatocytes, its presence in bile suggests transcytosis via the biliary epithelium.

Monte carlo simulation of the electron paramagnetic resonance spectrum displayed by copper ceruloplasmin at 77 K

Human ceruloplasmin contains several intrinsic copper ions, some of them being paramagnetic. The presence of two classes of paramagnetic Cu2+ ions (type I and type II) has been suggested, the two types having significantly different spectroscopic features. However, there are conflicting reports both on the ratio of the number of type-I to type-II Cu2+ and on their EPR spectroscopic parameters. By using a Monte Carlo method we obtained the best fit of the experimental spectrum with a synthesized composite spectrum generated by two type-I Cu2+ (type Ia and type Ib, possessing slightly different spin Hamiltonian parameters) and by one type-II Cu2+. Correspondingly, the EPR spectroscopic parameter (viz. theg-tensors, hyperfine couplings, line widths and molar fractions) together with their accuracies were determined.

Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.

A molecular model for the structure of human ceruloplasmin is proposed that is based on the determination of the complete amino acid sequence, studies of the products of limited proteolytic cleavage, calculations of the hydrophilic/hydrophobic character (hydropathy profile), and predictions of the local secondary structure. This multicopper oxidase (Mr approximately 132,000) consists of a single polypeptide chain (1046 amino acid residues) with four attached glucosamine oligosaccharides. Computer-assisted statistical analysis of the internal repetition in the amino acid sequence confirms that the entire polypeptide chain is divided into three contiguous homology units, each containing about 350 amino acid residues. Each homology unit is subdivided into three domains, designated A1, A2, and B, that differ in structure and probably in function. Calculations of the hydropathy profile and predictions of the secondary structure support a molecular model based on internal repetition of three homology units and help to identify characteristic features of the interdomain junctions. The alignment scores for internal duplication of pairings of the three homology units of ceruloplasmin exceed the scores yet reported for contiguous internal duplication of any other protein. This highly significant evidence for intragenic repetition suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes coding for a primordial copper oxidase.

Formation of a reactive iminium derivative by enzymatic and chemical oxidations of 16-O-acetylvindoline.

16-O- Acetylvindoline (1a) was oxidatively transformed into an iminium derivative (2a) by copper oxidases (laccase and human ceruloplasmin), an unknown enzyme system(s) of Streptomyces griseus, and the chemical oxidizing agent 2,3-dichloro-5,6- dicyano -1,4-benzoquinone ( DDQ ). The iminium derivative (2a) was isolated from enzymatic and chemical oxidation mixtures and was identified by spectral and chemical techniques. Reduction of the iminium compound with sodium borodeuteride provided monodeuterated 16-O- acetylvindoline (1b) as the sole product. Mass spectral analysis indicated that the deuterium atom was introduced into position C-3 of the piperidine portion of the alkaloid structure. The location and stereochemistry of the deuterium atom were confirmed by high-field 1H and 2H NMR analyses of the deuterated product to be in the 2H alpha orientation. Hydrolysis of the 16-O-acetyl functional group from the iminium derivative (2a) resulted in the production of a previously identified dimer (5), which forms by intramolecular etherification through the reactive enamine (3). The iminium derivative (2a) reacts with cyanide to provide complex mixtures of products, one of which was identified by mass spectrometry as a cyanide addition product. The results confirm the existence of a reactive iminium intermediate formed by all of the biochemical and chemical systems examined.

Specific receptor for ceruloplasmin in membrane fragments from aortic and heart tissues.

Membrane fragments from aortic and heart tissues of immature chicks were observed to bind highly purified, 125I-labeled chick ceruloplasmin. The binding reaction exhibited a linear Scatchard plot for both tissues and showed for each an apparent dissociation constant (Kd) of about 10(-8) M. On the basis of Scatchard analyses, aorta contained 1.5 pmol of receptors/mg of membrane protein, whereas receptors in the membranes from heart tissue were at least 5 times more dense. The binding of chick ceruloplasmin to aorta membranes was trypsin sensitive and neuraminidase insensitive, and showed both saturation and reversibility. Various sialoglycoproteins in 500 molar excess had very little effect on the binding. The asialo derivatives of these proteins likewise did not inhibit the binding. Human ceruloplasmin was found to bind very weakly to the chick membranes. Asialo chick ceruloplasmin bound with the same efficacy as native chick ceruloplasmin. Heat-denatured chick ceruloplasmin, however, was very ineffectual in displacing native 125I-ceruloplasmin from the membranes. These studies provide the first evidence for a homologous membrane receptor for native ceruloplasmin in the plasma membranes of animal cells.

Single-chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule.

We have determined the amino acid sequence of the amino-terminal 67,000-dalton (67-kDa) fragment of human ceruloplasmin and have established overlapping sequences between the 67-kDa and 50-kDa fragments and between the 50-kDa and 19-kDa fragments. The 67-kDa fragment contains 480 amino acid residues and three glucosamine oligosaccharides. These results together with our previous sequence data for the 50-kDa and 19-kDa fragments complete the amino acid sequence of human ceruloplasmin. The polypeptide chain has a total of 1,046 amino acid residues (Mr 120,085) and has attachment sites for four glucosamine oligosaccharides; together these account for the total molecular mass of human ceruloplasmin (132 kDa). The sequence analysis of the peptides overlapping the fragments showed that one additional amino acid, arginine, is present between the 67-kDa and 50-kDa fragments, and another, lysine, is between the 50-kDa and 19-kDa fragments. Only two apparent sites of amino acid interchange have been identified in the polypeptide chain. Both involve a single-point interchange of glycine and lysine that would result in a difference in charge. The results of the complete sequence analysis verified that human ceruloplasmin is composed of a single polypeptide chain and that the subunit-like fragments are produced by proteolytic cleavage during purification (and possibly also in vivo).

Isolation and purification of ceruloplasmin in oculocutaneous albinism, Menkes' disease, Wilson's disease and pregnant women

A method is reported for isolation and purification of human ceruloplasmin and apoceruloplasmin from serum. It involves a rapid and mild procedure by ion exchange chromatography on DEAE-Sephacel using a pH and ionic strength concave gradient. It was applied to serum of patients with oculocutaneous albinism, Wilson's disease, Menkes' disease and pregnant women. The ceruloplasmin obtained by this method is undegraded, and homogeneous by physico-chemical and immunochemical analysis.

Separation of limited tryptic fragments of human ceruloplasmin by gel-permeation high-performance liquid chromatography

Limited tryptic proteolysis of human ceruloplasmin rapidly produces several large, protease-resistant fragments, suggesting that the molecule consists of several domains. In order to locate the sites of proteolytic cleavage in the whole molecule, we used gel-permeation high-performance liquid chromatography to determine the optimum conditions for fragment separation. Using a buffer containing 8 M urea, the 67,000-daltons tryptic fragment from single-chain ceruloplasmin was isolated in a sufficiently pure state for amino acid sequence analysis to determine its location in the uncleaved molecule. These results have been used in conjunction with amino acid sequence data to develop a schematic model of the domain structure of human ceruloplasmin.

Identification of the thiol groups in human ceruloplasmin.

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.

Identification of the thiol groups in human ceruloplasmin

Identification of the thiol groups in human ceruloplasmin

Evidence for a single-chain structure of native human ceruloplasmin using sodium dodecyl sulfate-polyacrylamide-crossed immunoelectrophoresis

A simple method is described for the analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with crossed immunoelectrophoresis. The technique allows the identification of specific proteins and estimation of their molecular weights after the electrophoresis of complex mixtures such as blood plasma. Human ceruloplasmin was estimated to have a molecular weight of 128,000 using this method. Reduction of plasma with 2-mercaptoethanol in the presence of protease inhibitors did not result in dissociation of ceruloplasmin into detectable smaller subunits, suggesting that native ceruloplasmin is composed of a single polypeptide chain.

Cyro-vibrational spectroscopy of blue copper proteins

Resonance Raman (RR) and Fourier transform infrared (FTIR) spectra at 12K have been obtained for Pseudomonas aeruginosa azurin, spinach plastocyanin, stellacyanin, and tree laccase. The temperature dependence of the azurin, plastocyanin, and stellacyanin spectra have been recorded as have the RR excitation profiles at 12 K. Room temperature RR spectra have been obtained for azurins from Alcaligenes fecalis, Alcaligenes sp., Bortadella pretussis and Bortadella bronchiseptica; bean plastocyanin; fungal laccase, human ceruloplasmin; and zucchini squash ascorbate squash ascorbate oxidase. Isotope studies employing 63Cu/ 65Cu and H/D substitution have been performed on the azurins from Ps. aeruginosa, Alc. fecalis, and Alc. sp. Principal conclusion include the following; The intense RR modes near 400 cm −1 include internal ligand deformations and the Cu-S(cys) stretch, rather than the Cu-S(cys) stretch and Cu-N(his-Im) stretches as previously supposed. The Cu-N(his-Im) stretches are assignable to the ubiquitous feature near 265 cm −1,consistent with the frequencies of similar motions in other proteins and in model complexes. Spinach plastocyanin exhibits a frequency shift of 14 cm −1 in its cysteine CS stretching frequency ( ca. 750 cm −1) upon freezing of the protein solution, suggesting that extra-protein forces (e.g.,solvent structure, crystallization, or substrate binding.) can influence the conformation of the active site. Above the freezing point of the solvent the plastocyanin RR modes are unusually broad, suggesting either extremely facile due to thermally accessible conformations in the high-temperature form of the active site. No selective enhancement of either the strong or weak RR modes is observed in the S(cys)→ Cu charge transfer excitation profiles of azurin, plastocyanin, or stellacyanin at 12 K. The azurin species other than Ps. aeruginosa exhibit an ‘extra» strong RR peak near 400 cm −1 which is, however, seen to be related to an unresolved shoulder in the 12 K Ps. aeruginosa spectrum. It is therefore unnecessary to invoke higher coordination numbers than four for copper to explain the RR spectra of the azurins other than Ps. aeruginosa. RR peaks appear in the azurin spectrum below 200 K which may be due to methionine C-S stretching modes and Cu-S(met) stretch or methionine C-S-C angle bend. If these low-temperature features are indeed due to such motions,methionine must be closely coordinated to copper in azurin at low temperature. The RR spectra are consistent with a monotonic relationship between the force constant of the Cu-S(cys)bond and the energy of the ligand-field transitions of the various proteins. The isotope studies lead to tentative identification of the RR modes which contain significant contributions from M-L stretches and internal histidine motions. The results provide an initial basis for reliable structural interpretation of the RR spectra of the blue copper proteins.

Interactions of Purified Human Ceruloplasmin withLathyrus odoratus, Lern culinarisandCanavalia ensiformisLectins

Human ceruloplasmin was isolated from normal serum by fractional polyethylene glycol precipitation and subsequent ion exchange chromatography. The molecular mass determined by ultracentrifugation was 132 kDa and an optical density ratio (A610/A280) of 0.046 was observed in the preparation. Immunoelectrophoresis, agarose-gel electrophoresis and N-terminal amino-acid sequence analyses showed that the ceruloplasmin preparation was highly purified, while dodecyl sulphate polyacrylamide-gel electrophoresis indicated some degradation of the protein. Affinity chromatography showed that only a fraction of ceruloplasmin was bound to Lens culinaris or Lathyrus odoratus lectin-Sepharose, whereas nearly all the protein was bound to Canavalia ensiformis lectin-Sepharose. The carbohydrate composition of the ceruloplasmin fractions was analysed. It is suggested that fucose might be a determinant for the microheterogeneity in the carbohydrate chains of ceruloplasmin.

Internal triplication in the structure of human ceruloplasmin.

Amino acid sequence analysis of the 67,000-dalton (67-kDal) fragment that is the amino-terminal half of human ceruloplasmin has revealed internal triplication in the primary structure of the entire molecule. This is illustrated by comparison of 620 residues representing homologous domains of the 67-kDal fragment and of the 50-kDal and 19-kDal fragments that together comprise the carboxyl-terminal half of the molecule. The polypeptide chain is divided into three covalently linked homologous segments, each of about 340 residues. All three homology units have about 30% identity in sequence, and each pair exhibits at least 40% identity. The statistical significance of the 3-fold internal duplication was established by computerized analysis of the sequence. These results and studies of the sites of limited proteolytic cleavage support a model for the ceruloplasmin molecule consisting of an alternating structure of six domains of two different kinds (or possibly nine domains of three kinds). The 3-fold internal homology suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes.

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)

Microbial Transformations of Natural Antitumor Agents XVIII: Conversions of Vindoline with Copper Oxidases

Vindoline occurs structurally intact in the clinically important Vinca alkaloids vinblastine and vincristine. It is oxidized by human ceruloplasmin and fungal and plant laccases into a reactive intermediate which undergoes intramolecular cyclization to an enamine which ultimately dimerizes. Transformations of vindoline by these copper oxidases are enhanced when enzyme incubations are performed with cofactors such as chlorpromazine. The role of copper oxidases in alkaloid metabolic interconversions and the possible implications of these reactions in Vinca alkaloid toxicity are discussed.