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.     

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.     

Degradation and inactivation of ceruloplasmin and haptoglobin by rat liver lysosomes and some other proteinases

Lysosomal fraction was isolated from rat liver by density gradient centrifugation after pervious loading of lysosomes in vivo with Triton WR-1339. Tritosome preparations were incubated at 37 degrees C and pH 5 for 24 hr with purified human ceruloplasmin or haptoglobin. After this period approximately 20% of total alpha amino nitrogen was released from ceruloplasmin and over 40% from haptoglobin. This was accompanied by loss of peroxidase activity of haptoglobin (in complex with haemoglobin), while enzymatic activity of ceruloplasmin remained unaltered. Removal of sialic acid by neuraminidase had no effect on digestion of ceruloplasmin by rat liver tritosomes. Both glycoproteins were resistant to horse leucocyte proteinases and pancreatic eleastase but were easily inactivated by trypsin and chymotrypsin.     

Purification of Rat Ceruloplasmin: Characterization and Comparison with Human Ceruloplasmin

Rat ceruloplasmin was purified by a three-step column chromatography procedure, utilizing DEAE-Sepharose, Sepharose CL-6B, and CM-Sephadex A50 columns. The molecular weight of rat ceruloplasmin determined by a molecular sieve column was 124,000 daltons. An optical density ratio (610 nm/280 nm) of 0.051 and a molar extinction coefficient of 8600 were obtained. A decrease in lysine in rat ceruloplasmin compared with human ceruloplasmin could account for its reduced anodal mobility. Other differences in the amino acid sequence of the rat ceruloplasmin included an increase in methionine and cystine/cysteine, and a decrease in histidine, tyrosine and tryptophan.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

Ceruloplasmin carries the anionic glycan oligo/poly alpha2,8 deaminoneuraminic acid

Oligo/poly alpha2,8 deaminoneuraminic acid (KDN), a unique posttranslational protein modification, was found on megalin and a not yet characterized 150 kDa glycoprotein. We purified this glycoprotein from rat testis and identified it as ceruloplasmin. Furthermore, immunoprecipitated ceruloplasmin from rat thymus, ovary, blood serum, and postnatal day 2 but not adult lung and brain was immunoreactive for oligo/poly alpha2,8 KDN. The immunoreactivity for oligo/poly alpha2,8 KDN on purified serum ceruloplasmin was abolished by N-glycosidase F treatment but not by beta-elimination, indicating that it is present on N-glycosidically linked oligosaccharides. However, the copper binding activity of ceruloplasmin was independent of the presence of the anionic glycan. By immunohistochemistry, ceruloplasmin was detectable in histologically defined regions in rat ovary, thymus, and spleen. Likewise, by RT-PCR, ceruloplasmin expression was found in various non-hepatic rat tissues and showed a developmentally regulated pattern. Thus, ceruloplasmin, in addition to megalin, represents a glycoprotein carrying oligo/poly alpha2,8 KDN.     

Glycosyl phosphatidylinositol-anchored ceruloplasmin is expressed by rat Sertoli cells and is concentrated in detergent-insoluble membrane fractions.

The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.     

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.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications

Human liver fatty acid binding protein (L-FABP) cDNA clones were identified in a liver cDNA library. The two longest clones were completely sequenced. The nucleotide sequence predicts a protein of 127 amino acid residues. Identity of the clones was confirmed by limited amino acid sequence analysis of purified human L-FABP peptides and Edman degradation of radiolabeled in vitro translated FABP. Statistical analysis of the amino acid and mRNA sequences of human L-FABP, rat L-FABP, rat intestinal (I-) FABP, and mouse 422 protein indicates that the human and rat L-FABPs are highly homologous and that L-FABP and I-FABP diverged a long time ago (approximately 650-690 million years ago), although they are more closely related to each other than either of them is to 422 protein. Secondary structure predictions from the primary sequence of human and rat L-FABP reveal a region (residues 12-30) that might be the putative fatty acid binding domain of the two L-FABPs. Knowledge of the primary amino acid sequence of L-FABP and possible functional domains will be pivotal in further defining and understanding the mechanism of ligand binding and transfer by this protein.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases.

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.     

Mapping of the ceruloplasmin gene on human and laboratory mouse chromosomes by direct in situ hybridization

Mapping of ceruloplasmin gene in human and mouse chromosomes was carried out using the cloned fragments of rat chromosomal ceruloplasmin gene and of ceruloplasmin cDNA as specific hybridization probes. DNA probes were nick-translated with 125I-dCTP up to the high specific capacity. The number of silver grains as well as their distribution along the differentially stained chromosomes were analyzed in 120 metaphase plates from bone marrow cells of laboratory mice and in 181 plates from human lymphocyte cultures. The most probable localization of human ceruloplasmin gene is centromeric region q11-13 of chromosome 15(14?). In laboratory mice ceruloplasmin gene is assigned to the euchromatic part of D-disc of chromosome 9. The possible causes for gene synteny in laboratory mouse and in man as well as its evolutionary implication are discussed.     

Identification of catalytically important amino acids in human ceruloplasmin by site-directed mutagenesis

The involvement of amino acid residues previously proposed on the basis of structural data to have roles in the ferroxidase and diamine oxidase activities of human ceruloplasmin was investigated. Variants of human ceruloplasmin, in which residues proposed to be involved in electron transfer and/or iron-binding had been altered by site-directed mutagenesis, were expressed in HEK293 cells. E633A and E597A/H602A variants exhibited reduction in both activities by 50–60% compared to recombinant wild-type ceruloplasmin. The variant E935A/H940A had reduced ferroxidase activity (50%) but unaltered diamine oxidase activity, whereas the variant E971A exhibited enhanced diamine oxidase activity. For the L329M variant, both activities were identical to those of wild-type ceruloplasmin.     

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.     

Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.