[Properties of silver-containing rat ceruloplasmin]. / Svoistva tseruloplazmina krysy, soderzhashchego serebro.

Ceruloplasmin (Cp) was isolated from the sera of albino rats fed with silver nitrate (60 mg/kg of body weight). The oxidase activity of the enzyme was sharply decreased, while its concentration in the blood (as assayed immunologically) was slightly lower than in controls. The drop in the oxidase activity was caused by the replacement of several coppers by silver ions in the Cp molecule. Ag-Cp contained about four silver atoms per 1 mole of protein, its spectrum lacking maxima at 450 and 610 nm that are typical of normal Cp. When subjected to PAAG electrophoresis, Ag-Cp displayed two bands, one of which (Ag-Cp2) had the anodic mobility of normal Cp. The other band (Ag-CpI) migrated at a slower rate. Both bands were separately subjected to SDS-PAAG electrophoresis which revealed the dissimilarities among the proteolytic fragment patterns of Ag-CpI, Ag-Cp2 and normal Cp. Both Ag-CpI and Ag-Cp2 contained peculiar fragments produced by spontaneous limited proteolysis of the native molecule. The binding of silver ions by Cp seems to alter significantly the molecule conformation, which may cause the exposure of new peptide bonds susceptible to proteolytic attack. Cp seems to participate in the binding and detoxication of heavy metals in mammals.

[Etiologic significance of hereditary deficiency of proteinase alpha1-inhibitor in the formation of respiratory diseases]. / Etiologicheskoe znachenie nasledstvennogo defitsita alpha1-ingibitora proteaz v formirovanii zabolevanii organov dykhaniia.

A study was made of the content of protease alpha 1-inhibitor and of the phenotyping of protease alpha 1-inhibitor subtypes in 666 patients with different chronic non-specific pulmonary diseases. It is concluded that pronounced deficiency of protease alpha 1-inhibitor is of importance in the formation of primary emphysema, chronic obstructive bronchitis and bronchial asthma. The clinical characteristics of obstructive pulmonary diseases marked by protease alpha 1-inhibitor deficiency have been investigated.

[Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes]. / Sintez i sekretsiia tseruloplazmina izolirovannymi gepatotsitami krysy.

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.

[Molecular defect of ceruloplasmin synthesis and maturation in Wilson-Konovalov disease]. / Molekuliarnyi defekt sinteza i sozrevaniia tseruloplasmina pri bolezni Vil'sona-Konovalova.

The radioimmunochemical study of ceruloplasmin-synthesizing polyribosomes was carried out using bioptic liver specimens obtained from fourteen homozygous patients with hepatolenticular degeneration (Wilson--Konovalov disease) and from eight control patients with various non-hereditary diseases. The measurement of binding of 125I-antibodies to the nascent polysome-bound ceruloplasmin chains demonstrated that in control patients this protein is only synthesized on membrane-bound polysomes, while free polysomes do not contribute to the synthesis of ceruloplasmin. The majority of homozygous carriers of Wilson--Konovalov mutation (eleven of fourteen) are characterized by the involvement of free, rather than membrane-bound polysomes, in the synthesis of ceruloplasmin. This shift of ceruloplasmin synthesis from membrane-bound to free polysomes seems to be accompanied by disturbances in the cotranslational proteolytic maturation of ceruloplasmin from its biosynthetic precursors. As a result of this defect, a putative of ceruloplasmin (preproceruloplasmin) was detected in the content of Golgi complex as well as in the serum of homozygous patients. This precursor of a molecular weight 84,000 was found neither in Golgi complex, nor in the serum of control subjects.

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).

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.

[Molecular mechanisms of the control of ceruloplasmin synthesis by estradiol]. / Molekuliarnye mekhanizmy regulirovaniia sinteza tseruloplazmina pod deistviem estadiola.

Mechanisms of the effect of estradiol on the biosynthesis of ceruloplasmin (CP) in rat liver were studied. The radioimmunological tests revealed that estradiol caused an increase of CP concentration in plasma. The about two-fold increase of plasma CP level was accompanied by the proportional increase of the content of CP nascent chains in membrane-bound polysomes of liver. Cell-free translation of free and membrane-bound polysomes revealed that there is a two-fold increase in the relative rate of CP synthesis by membrane-bound polysomes from estradiol-treated rats, while free polysomes did not contribute to CP synthesis. The similar increase of the rate of CP synthesis was found also when RNA preparations from membrane-bound polysomes and from post-polysomal supernatant were translated in a heterologous cell-free system. Studies on the kinetics of hybridization with a specific cDNA probe showed that the increase in the rate of CP synthesis correlates to the increase of the steady-state level of CP mRNA in both membrane-bound polysomes and post-polysomal supernatant. A conclusion is made that the induction of CP by estradiol is related to the accumulation of CP-mRNA in liver cells.

[Estradiol-induced formation of the polyribosomal complex synthesizing ceruloplasmin in rats]. / Induktsiia obrazovaniia pod vliianiem setradiola poliribosomnogo kompleksa, sinteziruiushchego tseruloplazmin u krys.

The effects of estradiol on the oxidase activity of ceruloplasmin (CP) in rat serum and on the relative content of nascent CP chains in rat liver polyribosomes were studied. Using radioimmunological methods, it was shown that a 2--3-fold increase of the oxidase activity of CP in the blood induced by estradiol is accompanied by an increase in the amount of nascent CP chains in the polyribosomes. On the 5th, 8th, 10th and 11th post-injection days the binding of specific anti-CP 125I-immunoglobulins to the polyribosomes is increased by 17, 28, 70 and 95%, respectively. The values of sedimentation coefficients for the CP-synthesizing polyribosomes in the control and under estradiol induction are identical and make up to 425--430S. It is concluded that the increase of the oxidase activity of CP in the serum is due to stimulation of the de novo synthesis of CP rather than to the activation of the protein molecules.

Physicochemical properties and isoenzyme composition of hexokinase from normal and malignant human tissues.

The activity of hexokinase was studied in several normal and malignant human tissues. The enzyme activity in tumors was significantly higher. Isoenzyme studies on normal gastric mucosa and stomach cancer extracts showed that malignancy is accompanied by a "simplification" of the hexokinase isoenzyme pattern due to "deletion" of the slowest isoenzyme. Preparative polyacrylamide gel electrophoreis was used to isolate hexokinase isoenzymes from normal and malignant tissues. Tumor hexokinase isoenzymes displayed an increased affinity to glucose when compared to the corresponding normal prototypes (Km/glucose, 10(-6) M and 10(-5) M, respectively; Km = Michaelis constant). The molecular weights, subunit composition, and peptide patterns were identical for corresponding isoenzyme pairs from normal and tumor tissues.

[Tumorous origin of the hexokinase in human biological fluids]. / Ob opukholevom proiskhozhdenii geksokinazy v biologicheskikh zhidkostiakh cheloveka.

Hexokinase of the endometrium and gastric mucosa is represented by 5 isoenzymes. The "simplification" of HK isoenzyme spectrum is characteristic of cancer tissue. So, in gastric cancer there is a disappearance of the "slowest" isoenzyme, while in malignant endometrium the "fastest" one was absent. Hexokinase isoenzymes of the serum were identical to those in the tumors in question, that indicates the tumor origin of the body fluid hexokinase. The latter was not observed in normal body fluids. The isoenzymic composition of hexokinase in uterine fibromyoma did not differ from that in normal tissues. If hexokinase appeared in the serum of these patients, its isoenzymic composition was similar to that in the normal uterus. The study on the hexokinase isoenzyme composition may be a valuable adjunct in establishing the differential diagnosis between benign and malignant tumors.

[Identification of hexokinase synthesizing polyribosomes in human normal and tumor tissues]. / Identifikatsiia poliribosom, sinteziruiushchikh geksokinazy, v normal'nykh i opukholevykh tkaniakh cheloveka

Comparative study of concentrations of growing hexokinase subunits polypeptide chains in the polyribosomes from normal and tumour human stomach tissue is studied using specific antiserum to hexokinase isoenzyme from human stomach tumour tissue. It is shown by two different methods (chromatography on Sepharose 46 and sucrose concentration gradient analysis) that the content of hexokinase polypeptides in stomach tumour is several times higher than in homologous normal tissue. The acta obtained show that the high activity of hexokinase in tumour tissues is due to the increased synthesis rate of the enzyme.

[Physico-chemical and kinetic properties of hexokinase isoenzymes from human normal and tumor tissues]. / Fiziko-khimicheskie i kineticheskie svoistva izofermentov geksokinazy iz normal'noi i opukholevoi tkani cheloveka

Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000.

[Change in cell membrane permeability, and composition and properties of hexokinase during induced carcinogenesis]. / Izmenenie pronitsaemosti kletochnykh membran, sostava i svoistv geksokinazy v protsesse indutsirovannogo kantserogeneza

The isoenzyme hexokinase (HK) spectrum from normal rat large intestinal mucosa consisted of 3 isoenzymes. In tumours of this localization induced by 1,2-dimethylhydrazine there proved to be a lack or marked decrease in the most rapid anodic isoenzyme. Only one HK isoenzyme was found in the metastases. Km (glucose) for tumour HK was 2--3 times lower than for normal intestinal HK; the HK activity was detected in the serum from the 1st month of the carcinogenic administration, and by the 5th month it was found in 80% of the tumour-bearing animals. No serum HK activity was ever found in control rats.