Isolation and partial characterization of molecular forms of ceruloplasmin from human bile.

Highly purified ceruloplasmin (CP) was isolated from human bile using affinity chromatography. Biliary CP is represented by two molecular species. One of those is identical to oxidase CP from normal human serum while the other is analogous to oxidase-lacking CP specific for the serum of the carriers of Wilson's mutation with respect to immunological specificity, electrophoretical mobility and molecular mass of the large fragments from spontaneous proteolysis.

[Interaction of molecular forms of ceruloplasmin with specific membrane receptors from healthy persons and patients with hepatolenticular degeneration]. / Vzaimodeistvie molekuliarnykh form tseruloplazmina so spetsificheskimi retseptorami membran éritrotsitov zdorovykh liudei i bol'nykh gepatolentikuliarnoi degeneratsiei.

The ceruloplasmin (CP) interaction with the specific receptor localized on the erythrocyte membrane of healthy donors and of patients with hepatolenticular degeneration (HLD) was studied by using Scatchard analysis. It was found that in patients with the Wilson's disease the CP receptor is unaffected by the pathological process. The molecular mass of the erythrocyte receptor of CP is about 130 kDa. The CP binding to the specific receptor is followed by limited proteolysis of the latter. No CP binding to the receptor occurs in the presence of serine protease inhibitors. The CP-like protein previously detected in the sera of Wilson gene carriers can specifically interact with the CP receptor.

[Various properties of the ceruloplasmin receptor, isolated from human erythrocyte membranes]. / Nekotorye svoistva retseptora tseruloplazmina, vydelennogo iz membran éritrotsitov cheloveka.

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.

[Biosynthesis of ceruloplasmin in various rat organs]. / Biosintez tseruloplazmina v razlichnykh organakh krysy.

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.

[Expression of ceruloplasmin gene in various organs of the rat]. / Ekspressiia gena tseruloplazmina v razlichnykh organakh krysy.

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.

Molecular forms of ceruloplasmin in hepatolenticular degeneration and their interaction with human erythrocyte ceruloplasmin receptor.

Immunochemical methods were used to show that the sera of homozygous and heterozygous carriers of the Wilsonian gene contain, together with normal ceruloplasmin (CP), a CP-like protein that differs from CP in its enzymatic, immunological, and physicochemical properties. The CP-like protein was isolated from the sera of patients with hepatolenticular degeneration (HLD) by means of affinity chromatography, and monospecific antibodies to this protein were obtained. The presence of an 80 kDa immunoreactive polypeptide specific to the CP-like protein was demonstrated by immunoblotting with antibodies to normal CP and monospecific antibodies to the CP-like protein. Analysis of the ratios of the molecular forms of CP in homozygous and heterozygous carriers of the Wilsonian gene indicated that this ratio reflects the dosage of the mutant gene. The kinetic parameters of the interaction of these proteins with the CP-specific receptor on the erythrocyte membranes from healthy individuals and from patients with Wilson's disease (HLD) were determined. The CP receptor on erythrocyte membranes in HLD patients is not altered and its interaction with normal CP has the same kinetic parameters as the binding of normal CP to the erythrocyte receptors from healthy individuals. The CP-like protein also retains the ability to bind to the CP-specific receptor but the ligand-receptor complex is less stable than in the case of normal CP. The possible mechanism of the molecular heterogeneity of CP in the Wilsonian mutation is discussed.

[Molecular genetics of human monogenic diseases]. / Molekuliarnaia genetika monogennykh boleznei cheloveka.

The review deals with the analysis of the molecular basis of cytoplasmic genetic determinants and their contribution to inherited disease, as well as with the primary genetic defects underlying some single gene human disorders, such as hepatolenticular degeneration, alpha 1-antitrypsin deficiency, familial hypercholesterolemia, and cystic fibrosis. The results of molecular genetic studies of inherited diseases have been applied to the antenatal and preclinical diagnosis at the levels of mutant genes and anomalous proteins, gene products.

[Experience in heterozygote detection in Wilson-Konovalov mutation based on the study of molecular forms of ceruloplasmin]. / Opyt vyiavleniia geterozigot po mutatsii Vil'sona-Konovalova na osnove issledovaniia molekuliarnykh form tseruloplazmina.

A specific novel molecular form of ceruloplasmin (CP) was detected in the sera of Wilson's disease patients and their closest relatives using two-dimensional cross-immunoelectrophoresis. This protein shares some antigenic properties with normal CP but is not completely identical to the latter. Besides, anomalous CP has no oxidase activity of normal CP and differs from it in electrophoretic mobility in agarose and polyacrylamide gels. Anomalous CP was purified to homogeneity and monospecific antibodies to this protein were obtained. The quantitative analysis showed that the ratios of normal CP to anomalous CP in homo- and heterozygous carriers of the "Wilsonian" gene are reproducibly different and can be used as a diagnostic test allowing the differentiation between these groups.

[Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin]. / Spektral'nye issledovaniia mekhanizma oksidaznoi aktivnosti tseruloplazmina.

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.

Chromosomal localization of ceruloplasmin and transferrin genes in laboratory rats, mice and in man by hybridization with specific DNA probes.

Fragments of the natural rat ceruloplasmin (Cp) gene and cDNA copies of rat Cp and transferring (Tf) mRNAs highly labelled by nick translation with 125I-dCTP were used as specific probes for assignment of these genes to the metaphase chromosomes of rat, mouse and man by in situ hybridization. Both Cp and Tf genes were found to be syntenic in rodents, occupying with high probability the regions 9D and 9F1-3 in mice and 7q11-13 and 7q31-34 in rats respectively. The significant increase in silver grain count over chromosome 15 in rats after hybridization with both the Cp and Tf probes suggests the presence of a related pseudogene cluster on this particular chromosome and thus favours its partial homeology to chromosome 7. The localization of silver grains in metaphase chromosome of man indicates subregional assignment of the Tf gene to 3q21. Use of the rat Cp DNA probe does not indicate synteny of the Cp and Tf genes in man and suggests the existence of a related DNA sequence in 15q11-13. The potential and limitations of the in situ hybridization technique with heterologous DNA probes for gene mapping in mammalian species are discussed.

[Comparative study of ceruloplasmin biosynthesis in various cell-free systems]. / Sravnitel'noe izuchenie biosinteza tseruloplazmina v razlichnykh beskletochnykh sistemakh.

Some peculiarities of mRNA translation of ceruloplasmin (CP) from rat liver were investigated, using three cell-free protein biosynthesis systems (wheat embryo extracts, rabbit reticulocyte lysates and Zajdela ascite hepatoma extracts). It was shown that reticulocyte lysates and tumour cell extracts synthesize full-size CP mRNA translation products, whose molecular mass is close to that of mature CP molecules, i. e., 122-132 kD. Wheat embryo extracts synthesize the NH2-terminal fragment of the CP molecule (Mr = 84 kD). Addition of liver membrane fractions to wheat embryo extracts translating CP mRNA results in the reconstitution of proteolytic steps of CP maturation.

[Mapping of the ceruloplasmin gene on human and laboratory mouse chromosomes by direct in situ hybridization]. / Kartirovanie gena tseruloplazmina na khromosomakh cheloveka i laboratornykh myshei metodom priamoi gibridizatsii in situ.

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.

[Use of specific DNA probes for mapping the ceruloplasmin gene on rat chromosomes by direct in situ hybridization]. / Primenenie spetsificheskikh DNK-zondov dlia kartirovaniia gena tseruloplazmina na khromosomakh krysy metodom priamoi gibridizatsii in situ.

Specific DNA-probes representing the fragments of chromosomal ceruplasmin gene (lambda RCp-1, lambda RCp-2, lambda RCp-3) and its cDNA copy of the corresponding mRNA were heavily labelled with 125J dCTP (the specific activity of the probes varying from 1.5 X 10(7) to 3.4 X 10(8) dpm). These probes were used for in situ hybridization on metaphase chromosomes. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 653 metaphase plates from bone marrow cells of laboratory rats. The results of in situ hybridization were very similar for all four specific DNA-probes tested and allow to assign ceruplasmin gene to the q13 region of chromosome 7. The local increase of silver grain count over chromosome 15 was also registered and discussed.

Evidence that human ceruloplasmin molecule consists of homologous parts.

The products of spontaneous and induced proteolysis of human ceruloplasmin (Cp) were studied. Some physico-chemical properties of the six fragments with electrophoretically determined Mr 130,000 (F1), 110,000 (F2), 66,000 (F3), 48,000 (F4) 22,000 (F5) and 18,000 (F6) were compared. The amino acid compositions and N-terminal amino acid sequences coincide in F1-F5, but differ from those of F6. Limited tryptic proteolysis of Cp causes the accumulation of polypeptide fragment with Mr 22,000, the N-terminal primary structure of which is identical to that of F5 produced by spontaneous proteolysis. Electrophoretic fragments of Cp were extracted from polyacrylamide gel, treated with 125I and then studied by peptide mapping with subsequent radioautography. The comparison of the "finger prints" showed the identity of F1 to F2 and F3 and gross similarity between F4 and F1-F3. It also revealed similar peptides in F5 and F6 hydrolyzates and almost perfect matching of the F4 map to the map of F5 + F6 mixture. On the basis of the obtained data general principles of Cp molecular organization are discussed and intramolecular homology is suggested to be a feature of the protein.

Identification of ceruloplasmin messenger RNA sequences in heterogeneous nuclear RNA from rat liver.

The distribution of the sequences of ceruloplasmin mRNA in different fractions of heterogeneous nuclear RNA from rat liver was studied using cDNA transcripts of highly purified mRNA as hybridization probe. The content of ceruloplasmin mRNA sequences in poly(A)-containing and poly(A)-free subfractions of heterogeneous nuclear RNA is respectively 1 and 27 molecules per a hepatocyte. Heterogeneous nuclear RNA carrying the sequences of ceruloplasmin mRNA sedimented in sucrose gradients containing formamide, as a broad zone around the 56S peak. Denaturing electrophoresis followed by the transfer of RNA onto diabenzyloxymethyl paper and hybridization with [32P]-cDNA revealed multiple high molecular weight fractions of ceruloplasmin pre = mRNA (9.0, 6.6, 2.2 and 1.6 megadaltons) in the non-adenylated fraction of nuclear RNA and a single 1.1-1.2 megadalton zone in poly(A)-containing nuclear RNA, the latter being equal in size to the mature ceruloplasmin mRNA from liver polysomes.

Preproceruloplasmin is a primary product of cell-free translation of ceruloplasmin messenger RNA.

Biosynthesis of ceruloplasmin was studied in wheat germ extract programmed with polysomal RNA from rat liver. Optimal potassium concentration for the total protein-synthesizing activity and for the synthesis of immunoreactive ceruloplasmin was 96 and 186 mM respectively. 7-methylguanosine 5'-monophosphate caused two-fold inhibition of the cell-free synthesis of ceruloplasmin. Immunoprecipitated ceruloplasmin that was synthesized at optimal potassium concentration was a homogeneous polypeptide of a molecular weight about 84 kD. The addition of membrane fractions from rat liver to the incubation mixture caused the conversion of the 84 kD polypeptide into 80 kD and 65 kD polypeptides that are similar to proceruloplasmins synthesized in rat liver during in vivo pulse labelling. The suggestion is made that 84 kD polypeptide is a primary product of the translation of ceruloplasmin mRNA (preproceruloplasmin).

Highly purified ceruloplasmin messenger RNA from rat liver. Physico-chemical and functional characteristics.

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 . 10(6) daltons which is large enough to code for a putative precursor of ceruloplasmin (approximately 700 amino acid 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 (m7G5'ppp5'XmpAp). 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.

Intracellular distribution of rat-liver polyribosomes synthesizing coeruloplasmin.

Three independent approaches (binding of 125I-antibodies to polysomes, cell-free translation of polyadenylated mRNA in a wheat germ system and hybridization of RNA with a specific complementary DNA probe) were used to study the intracellular distribution of polysomes involved in the synthesis of coeruloplasmin as well as of coeruloplasmin mRNA sequences in rat liver. It was shown that only membrane-bound polysomes contain both nascent chains of coeruloplasmin and coeruloplasmin mRNA sequences. The size of coeruloplasmin polysomes is 16-18 monomers/mRNA molecule and their proportion is about 0.4-0.6% of total liver polysomes. The proportion of coeruloplasmin mRNA is 0.0025% of the total polysomal RNA and 0.29% of poly(A)-containing mRNA. These values correspond to coeruloplasmin mRNA concentration about 400-535 molecules per parenchymatous liver cell, 90% of those mRNA molecules were recovered from polysomes while the remaining 10% were from postpolysomal supernatant.