Genetic polymorphism of ceruloplasmin in the rat

The genetic heterogeneity of ceruloplasmin in serum was studied in the progeny of the (LEW X BN)F1 X (LEW X BN)F1 rats. The results of the statistical analysis showed that the Hbb and c loci were linked. However, the autosomal Ces gene was not linked to the Hbb or c loci. The Ces gene was expressed in normal Mendelian pattern and had two alleles that manifested a low (Cesl) or high (Cesh) level of the enzyme in serum. The Cesl gene was expressed as a dominant in the F1 generation. Two phenotypes CES-H and CES-L were found in the F2 rats; the females in the parental strain and hybrid had higher ceruloplasmin concentration than the male rats.     

Biosynthesis of ceruloplasmin in various rat organs

Ceruloplasmin (CP) biosynthesis in various organs of the rat was studied. It was found that the translation products of postmitochondrial extracts of various organs of the rat contain immunoreactive polypeptides of CP. In respect of proteolytic fragments formed during the digestion with staphylococcal protease V8, these polypeptides do not differ from one another. At the same time, in Golgi vesicles of the kidney, brain and liver the mature molecular forms of CP differ not only by their molecular properties, but also by the number of CP isoforms. For example, the organs which do not secrete CP, contain only one isoform of CP. The secreted form of CP was found to be tissue-nonspecific. The third molecular form of CP found in the liver has no counterparts in the other organs. Immunochemical analysis of mature forms of CP isolated from liver Golgi vesicles provided additional evidence in favour of molecular heterogeneity of CP secreted by the liver. The mechanism of production of multiple molecular forms of CP and their roles in copper metabolism are postulated.     

Isolation and physico-chemical properties of rat ceruloplasmin

Ceruloplasmin was isolated and purified from albino rat blood serum. Relative molecular mass of the protein is 130 000. Electrophoresis of the protein preparations leads to a formation of the apo-protein devoid of the oxidase activity and migrating slower than the holo-protein. Leucine was found to be the N-terminal amino acid of the ceruloplasmin polypeptide chain. The amino acid composition and carbohydrate content of the protein were determined. The tryptic peptide maps of rat ceruloplasmin were compared to those of human protein. The properties of rat and human ceruloplasmin are discussed with respect to copper metabolism in animal body as well as in normal humans and patients with Wilson's disease.     

An enzyme-linked immunoadsorbent assay for rat ceruloplasmin

A noncompetitive, colorimetric enzyme-linked immunoadsorbent assay (ELISA)† was developed for measuring rat ceruloplasmin in serum and in medium from culture hepatocytes. The assay utilized polystyrene immobilized antibody which bound ceruloplasmin which then bound biotinylated antibody. The biotinylated antibody-antigen complex was detected with strepavidin-alkaline phosphatase conjugate. Standard curves for rat ceruloplasmin were constructed in the range between 10 and 50 ng/mL. Increases of 10 ng produced an increase inA ₄₀₃ of more than 0.2. With this immunoassay, serum ceruloplasmin levels were found to average 35 mg Cp/dL in control rats and 87 mg/dL in rats with experimental inflammation. Liver parenchymal cells secreted 1.6μg Cp/5×10⁵ cells/24h. This ELISA assay for ceruloplasmin will facilitate studies on the regulation of ceruloplasmin synthesis and secretion in both intact rats and isolated hepatocytes.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes

The synthesis and secretion of ceruloplasmin (Cp) by isolated rat hepatocytes were investigated. Cp released by liver cells appeared to have properties similar to those of the blood-circulating protein, i. e. Mr, oxidase activity, immunological specificity and the peptide set of tryptic fingerprints. The polypeptides with Mr of 130,000, 65,000, 48,000 and 18,000 were revealed in Cp isolated from the incubation medium. These results suggest the susceptibility of the single-chain protein molecule (Mr 130,000) to limited proteolysis which is accomplished by the proteases released from the cells. When fresh serum was added to the incubation medium, the proteolytic degradation of Cp proceeded at a much slower rate, which led to an increase in the content of excreted polypeptides with Mr 130,000. The secretion was strongly diminished by the addition of colchicine to the medium. The time of Cp molecule synthesis on membrane-bound polyribosomes (3.5 min) was determined.     

Expression of ceruloplasmin gene in various organs of the rat

The contribution of different rat organs to the synthesis of ceruloplasmin (Cp) was studied. Dot hybridization with the use of the Cp cDNA probe revealed Cp mRNA sequences in RNA preparations from liver, heart, kidney as well as from different divisions of brain, the concentration of Cp mRNA sequences being maximal in the liver. Polyribosomes isolated from these organs effectively synthesized Cp in a cell-free system derived from rabbit reticulocytes. After in vivo pulse labeling, the newly formed radioactive Cp was detected in the membrane fractions from all these organs. The newly formed Cp was concentrated within the membranes of the Golgi complex of various organs where it was revealed by different immunochemical techniques. Experiments with isolation of the liver from the systemic circulation showed that the liver is the only organ secreting Cp into the blood stream. It was suggested that mammalian tissues contain at least two molecular forms of Cp, i. e., circulatory and intracellular ones.     

Detection of rat ceruloplasmin receptors using fluorescence microscopy and microdensitometry

Rat ceruloplasmin (rCp) has been labeled with the fluorophores fluorescein and rhodamine by using the isothiocyanate derivatives (FITC, RBITC). High p-phenylenediamine oxidase activity of the resulting conjugates was observed (70-90% of native activity). Polyacrylamide gel electrophoresis showed fluorescein-labeled rCp (FITC-rCp) had an increased mobility, while rhodamine-labeled rCp (RBITC-rCp) showed no increase in mobility when compared to native rCp. RBITC-rCp was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy to detect membrane binding. Film negatives of blood smears exposed under identical conditions were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound per unit area of the plasma membrane. With this technique, binding of rCp was observed to be saturable, reversible, and specific. Competition of the protein ligands superoxide dismutase, catalase, and unlabeled rCp against RBITC-rCp already bound on erythrocytes showed that only rCp could displace the bound RBITC-rCp with 32% specific binding being observed. Low levels of membrane binding were seen after the erythrocytes had been trypsin treated. Platelet binding was also detected and it was saturable, reversible, and trypsin sensitive. However, only 20% of the bound RBITC-rCp could be displaced by rCp. These studies demonstrate a versatile technique for detection and localization of Cp receptors.     

Enhancement of rat serum ceruloplasmin levels by exposure to hyperoxia

Exposure of rats to elevated oxygen tensions is a well-known method of producing enhanced levels of pulmonary antioxidant enzymes. Ceruloplasmin is a serum constituent which possesses scavenging activity toward oxygen radicals. Rats exposed either to continuous 85% oxygen for 7 days or to intermittent hyperbaric oxygen for up to 19 days developed not only enhanced lung antioxidant enzymes but also increased levels of serum ceruloplasmin. The latter did not appear to be merely an "acute phase reactant" as there was no change in total serum protein, plasma fibrinogen, serum -SH groups, sedimentation rate, or serum iron. Induction of ceruloplasmin may account for some of the anti-inflammatory activities of elevated oxygen tensions.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium

Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.     

The effect of ceruloplasmin on hepatocyte proliferation in the regenerating rat liver

The influence of ceruloplasmin on cell proliferation in regeneration liver of the rat has been studied. Ceruloplasmin stimulates cell proliferation in regeneration liver increasing functional activity of the mononuclear phagocytes.     

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.