Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.16.3.1 (ceruloplasmin)
 5,074 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Identification of the thiol groups in human ceruloplasmin. 684 89

A histidine-rich fragment, Cp F5, with a molecular weight of 18,650 was isolated from human ceruloplasmin. It consists of 159 amino acids and contains a possible copper-binding site. The sequence of the first 18 NH2-terminal residues of Cp F5 was determined by automated Edman degradation. Cp F5 was cleaved by cyanogen bromide to produce nine fragments of from 2 to 63 residues. The amino acid sequence of all of the cyanogen bromide fragments was investigated using automated and manual Edman degradation, the fragments being digested with trypsin, chymotrypsin, thermolysin, staphylococcal protease, and pepsin as appropriate. The results, in conjunction with the data on the tryptic peptides reported in the accompanying paper (Kingston, I.B., Kingston, B.L., and Putnam, F.L. (1980) J. Biol. Chem. 255, 2886-2896), establish the complete amino acid sequence of Cp F5.
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PMID:Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. I. Amino acid sequence of the cyanogen bromide peptides. 698 29

Concentrations of the following acute-phase reactants (APR) were determined in the sera of patients with benign and malignant ovarian tumors: haptoglobin, ceruloplasmin, sialic acid, seromucoid (in sulphosalicylic acid supernatant), and trypsin inhibitory capacity. In patients with ovarian carcinomas significant increase (p less than 0.001) in all measured APR parameters as compared to healthy women and women with benign tumors was found. Moreover, the levels of haptoglobin, ceruloplasmin and trypsin inhibitory capacity were statistically lower (p less than 0.001) in nonmalignant than in malignant ovarian disease. No distinct statistical difference was confirmed between healthy controls and the benign tumor group as well as between ovarian carcinomas of Stages I--III and Stage IV.
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PMID:Clinical usefulness of serum acute-phase reactants in patients with ovarian tumors. 729 Feb 69

The use of activated thiopropyl-Sepharose for simple and rapid isolation of thiol peptides from large proteins was investigated using ceruloplasmin (a copper protein of molecular weight 134,000 containing three cysteines and six disulphides) as a test case. Optimal results for the immobilization of the protein to the activated gel were obtained at pH 4.0 in the presence of 8 M urea and 0.05 M ethylenediaminetetraacetic acid. In these conditions 96% of the protein thiol groups were attached to the adsorbent. The immobilized protein was digested with either pepsin or trypsin. The liberated non-thiol peptides were eluted from the gel together with the protease after the digestion. After washing, the covalently attached thiol peptides were eluted in reducing buffer, desalted on the hydrophobic gel Sephadex LH-20 and carboxymethylated. The peptides were purified in a two-step procedure involving gel filtration on Sephadex G-25 and either column electrophoresis or ion-exchange chromatography. The two sets of peptides were derived from four different regions in the protein. They were 12-39 residues in length and together accounted for 152 residues. It is shown that the peptide chain was susceptible to proteolytic attack also close to the point of attachment (two residues away). One peptide with two thiol groups proved to be derived from an area containing one disulphide bridge in addition to cysteine. This bridge could be identified in a separate experiment where a second enzyme was used to release the disulphide peptide after the first digestion and washings.
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PMID:Covalent chromatography as a means of isolating thiol peptides from large proteins: application to human ceruloplasmin. 732 Jan 3

The yeast FET3 gene is required for high affinity iron transport (Askwith, C., Eide, D., Ho, A. V., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403-410). The gene has extensive sequence homology to the family of multi-copper oxidases. In this communication, we demonstrate that the gene product is a cell surface ferroxidase involved in iron transport. Cells that contain a functional FET3 gene product exhibited an iron-dependent non-mitochondrial increase in oxygen consumption. Comparison of the rate of iron oxidation to O2 consumption yielded an approximate value of 4:1, as predicted for a ferroxidase. Spheroplasts obtained from cells grown under low iron conditions also displayed an iron-dependent increase in O2 consumption. Treatment of spheroplasts with trypsin or affinity-purified antibodies directed against the putative external ferroxidase domain of Fet3 had no effect on basal O2 consumption but inhibited the iron-dependent increase in O2 consumption. Anti-peptide antibodies directed against the cytosolic domain of Fet3 had no effect on O2 consumption. These studies indicate that Fet3 is a plasma membrane ferroxidase required for high affinity iron uptake, in which the ferroxidase-containing domain is localized on the external cell surface.
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PMID:The FET3 gene product required for high affinity iron transport in yeast is a cell surface ferroxidase. 783 66

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.
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PMID:Characterization of a particulate pathway for copper in K562 cells. 813 Feb 71

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable production of AFR was detected, which was increased by a 10(-4) M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu2+ and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive interactions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo.
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PMID:An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins. 954 60

This study was mainly aimed to investigate the efficacy of trypsin:chymotrypsin to elicit anti-oxidant properties. In our earlier studies it was observed that the enzyme preparation exhibited an anti-inflammatory action as there was a remarkable reduction in oedema formation and tissue destruction. This led to further study on the amount of lipid peroxidation products formed and the levels of enzymatic and non-enzymatic anti-oxidants and relative trace element contents of copper, selenium, iron and zinc during administration of the enzyme preparation. Decreased formation of lipid peroxidation products was observed in treated group in comparison with the untreated group. Higher levels of enzymatic anti-oxidants mainly super oxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase and non-enzymatic antioxidant namely ceruloplasmin persisted for a longer period of time in the treated group than in the untreated group. No statistical significance was observed in non-enzymatic antioxidants viz. ascorbic acid and tocopherol levels in both the groups. Increased serum copper and selenium levels in the treated group could be related to higher levels of the ceruloplasmin and glutathione peroxidase observed in the treated group. The above studies support the finding that treatment with the enzyme preparation reduced tissue destruction leading to decreased formation of free radicals and subsequent effective scavenging of free radicals by the higher levels of enzymatic and non-enzymatic anti-oxidants.
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PMID:The efficacy of trypsin: chymotrypsin preparation in the reduction of oxidative damage during burn injury. 977 92

A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3.
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PMID:Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis. 1060 Jan 67

Using SDS-PAGE and MALDI-TOF mass spectrometry, we investigated the difference in the molecular structure between human and bovine ceruloplasmin. In both cases, we found that the protein is present in two majors forms of different molecular mass. The difference between human and bovine ceruloplasmin was more obvious when characterized by MALDI-TOF than with the SDS-PAGE analysis. Furthermore, we established that the N-glycoside content of both enzymes is dissimilar and that the N-glycosyl moieties are distributed in a distinctive fashion in two glycoproteins. Finally, it appeared that both proteins exhibited different cleavage patterns after treatment with trypsin. This study indicates that human and bovine ceruloplasmin differ not only in sugar composition but also in primary structure.
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PMID:Molecular characterization of human and bovine ceruloplasmin using MALDI-TOF mass spectrometry. 1168 10


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