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Biomedical subjects

E Regoeczi

Publications and source records attributed to E Regoeczi.

At least 55 records · Page 3Linked to original sources
PubMed1984

Synthesis and turnover of prothrombin during experimental inflammation in rats.

The response of prothrombin to inflammatory reactions was investigated in rats. Inflammation was induced by the administration of either subcutaneous turpentine or intraperitoneal endotoxin, and its effects were studied 24 h and 48 h later. Albumin and alpha 1-acute-phase globulin served as the controls. There were only insignificant changes in plasma prothrombin concentration during inflammation which contrasts sharply with a decrease in circulating albumin by approximately 25% and an increase in alpha 1-acute-phase globulin by 300-400%. These changes were paralleled by similar changes in the incorporation of [3H]lysine into these proteins during the incubation of liver slices from rats that had been pretreated with the phlogistic agents. Prothrombin catabolism, studied using 131I-prothrombin, was increased by approximately 20%; albumin turnover, studied simultaneously with 125I-albumin, was not significantly affected, though the capillary transfer rate of albumin was significantly elevated 48 h after the induction of inflammation. It is concluded that rat prothrombin is not an acute-phase protein.

Animals↗
PubMed1983

The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms.

Crude bacterial collagenase was chromatographed on DEAE-cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% w/v), which produced 70-80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion two gelatinase-dependent, cell-releasing mechanisms were identified: (a) DEAE-3 with ZnII peak 1 and (b) DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.

Animals↗
PubMed1983

Iodogen-catalyzed iodination of transferrin.

Transferrin (human, rabbit) labels at low efficiency (1%-10%) with 125I when reaction of 0.5-0.7 ng of I- (8-10 microCi) with 20 micrograms of the protein is catalyzed by iodogen in a constant volume of 0.1 ml. Microiodination by this technique was therefore analyzed with regard to the relative proportions of the reactants, oxidant requirement, and timing. In vials giving a reaction volume-to-active surface ratio of 0.88, efficiency was independent of the amount of iodogen in the range from 1 microgram to 15 micrograms, and prolongation of the reaction beyond 1 min failed to improve yields. In contrast, the amount of I- present was decisive. Butanol/NH4OH chromatograms of iodination reactions carried out with 0.6 ng or 20 ng of I- showed 3-4 radioactivity peaks, the relative proportions of which markedly depended on the amount of I- present originally. A link was established between labeling efficiency and chromatographic profile of the I- derivatives formed during oxidation. Dual-label experiments in rats showed that transferrin (20 micrograms) can be labeled using iodogen (1-5 micrograms, 1 min) to behave indistinguishably from its IC1-labeled counterpart. However, prolonged exposure to more oxidant progressively damaged the protein. The damage was independent of substituting I and it manifested itself in increased protein binding to the anion exchange resin, Dowex 1-X8. Over 99.5% of the labeled residues in iodotransferrin were mono- and diiodotyrosines (MIT, DIT). DIT content of the protein increased linearly with the number of I atoms substituted. At comparable levels of substitution, more label was present as MIT after using iodogen than after using IC1. Electrophoretic data are presented regarding homogeneity of the label as obtained after iodinating transferrin by different methods and to varying extents.

Animals↗
PubMed1982

Receptor-rich intracellular membrane vesicles transporting asialotransferrin and insulin in liver.

A wide range of receptors are located at the blood sinusoidal aspect of the hepatocyte plasma membrane. Many circulating ligands that bind to receptors on the cell surfaces are interiorized along two pathways. Asialoglycoproteins are transferred from the plasma membrane to lysosomes and degraded, whereas immunoglobulin A and bile acids are transported across the hepatocyte interior and released into bile. Asialotransferrin type 3 (ref. 6) follows a further pathway termed diacytosis. After binding to the asialoglycoprotein receptor, asialotransferrin is endocytosed and then returned to blood with a proportion of its carbohydrate side chains resialylated. We now describe in liver the properties of intracellular asialotransferrin-enclosing vesicles (diacytosomes) and show that they differ from Golgi, lysosome and plasma membrane fractions. Furthermore, we show that the asialoglycoprotein binding sites are located on the cytoplasmic (outer) surface of diacytosomes.

Animals↗
PubMed1982

Partial resialylation of human asialotransferrin type 3 in the rat.

After the injection of a small dose (1 micrograms/100 g of body weight) of 125I-labeled human asialotransferrin type 3 in rats, the radioactivity became rapidly associated with the liver. However, during the ensuing 12 hr a significant fraction of the dose returned to the circulation as protein-bound 125I. The protein released by the liver was indistinguishable by gel filtration from the original preparation and was precipitable by an antiserum to human transferrin. Nevertheless, it no longer bound to the immobilized Gal/GalN-specific lectin from rabbit liver. However, binding could be restored to a large extent by treatment with neuraminidase, indicating that the loss of binding was due to resialylation. Changes in the electrophoretic mobility of asialotransferrin released by the liver showed that resialylation was partial--i.e., it involved the attachment of two or three sialyl residues. From analysis by deconvolution of the plasma curve of partially resialylated asialotransferrin it was calculated that the liver "repaired" this way approximately one asialotransferrin molecule out of four. Plasma clearance of partially resialylated asialotransferrin was similar to that of nondesialylated transferrin.

Animals↗
PubMed1982

The structural heterogeneity of the carbohydrate moiety of desialylated human transferrin.

Human transferrin consists of a single chain polypeptide which supports two N-glycosidically linked glycans at sequons a and b. Glycopeptides were released from human transferrin by proteolytic digestion, desialylated by mild acid hydrolysis, and then isolated by chromatographic methods. The structures of the glycans located on each sequon were determined by a combination of analytical techniques including Smith degradation, permethylation, and enzymic degradation. Approximately 79% of the total glycan from sequon a was of the biantennary type as previously described by Dorland and his colleagues (FEBS Lett. 77, 15-20 (1977)). The remaining 21% consisted of a mixture of triantennary and tetraantennary glycans, each amounting to approximately 10% of the total glycan for this sequon. The triantennary structure resembled that described for the N-glycosidic triantennary glycans of bovine fetuin by Nilsson and his colleagues (J. Biol. Chem. 254, 4545-4553 (1979)). Of the tetraantennary glycan, approximately half of the structures were incomplete, i.e., one antenna terminated by N-acetylglucosamine. On sequon b, 81% of the glycan was biantennary, identical to those biantennary glycans of sequon a, and the reminder was triantennary, also of the fetuin type. The glycan structures and their locations on the polypeptide are related to the known subpopulations of human transferrin.

Amino Acids↗
PubMed1981

Subcellular distribution of human asialotransferrin type 3 in the rat liver.

A small quantity of 125I-labeled human asialotransferrin type 3 (2 to 4 microgram/100 g) was injected in intact rats and the distribution of the hepatic radioactivity analyzed by fractionation of liver homogenates on continuous sucrose density gradient. The ligand rapidly partitioned between plasma membrane and the interior of the cell at an approximate ratio of 1 to 4. The ratio remained constant between 3 min and 1 h. Intracellular 125I was encapsulated in a particle that was of a median equilibrium density of 1.11 (1.109 to 1.114) g/cm3 at 20 degrees C. The ligand recovered from the particles showed no sign of proteolytic digestion and was bound by the immobilized asialoglycoprotein-binding lectin from rabbit liver. The electron microscopic appearance of the subfractions containing of the entrapped ligand closely resembled that of an intermediate Golgi preparation. Various attempts were made to separate the ligand-containing particles from sialyltransferase and phosphodiesterase I activities, but complete separation could not be accomplished. 125I-Asialoorosomucoid studied in the same quantities and under the same conditions as asialotransferrin, yielded a subcellular distribution which was distinct from that of asialotransferrin type 3. Increasing the dose of asialotransferrin, to a level at which rapid catabolism of this asialoprotein occurs, profoundly changed the subcellular distribution of radioactivity. The subcellular distribution thus obtained was comparable with that found for asialoorosomucoid. These findings suggest that asialotransferrin type 3 is associated with different intracellular vehicles (different endosomes?) depending on whether the protein is simply diacytosed or is en route to lysosomes.

Animals↗
PubMed1981

Diacytosis of human asialotransferrin type 3 by isolated rat hepatocytes.

In suspensions of freshly isolated hepatocytes, asialotransferrin type 3 became rapidly bound by the asialoglycoprotein-binding hepatic lectin. Suspended hepatocytes, just as the liver of intact animals, catabolized asialotransferrin in a concentration-dependent manner. At low asialotransferrin concentrations (0.4-1 nM), the fraction of labeled protein degraded was much smaller than found with comparable concentrations of asialofetuin in an earlier study (Tolleshaug, H., Berg, T., Nilsson, M., and Norum, K. R. (1977) Biochim. Biophys. Acta 499, 73-84). The fraction of endocytosed asialotransferrin that was degraded, could, however, be substantially increased by raising the concentration of asialotransferrin the medium. Release studies using a chelating agent or competitive inhibitors of the binding reaction showed that at low asialotransferrin concentrations, hepatocytes exocytose the preponderance of the intracellular asialotransferrin with a half-life of approximately 20 min. This novel observation raises the possibility that lysosomal homing of an endosome transporting asialoglycoprotein requires an intracellular target signal.

Animals↗
PubMed1981

Binding of asialotransferrins by purified rat liver plasma membranes.

Interaction of four different asialotransferrins (human types 1, 2, and 3, and rabbit asialotransferrin) with purified plasma membranes from the rat liver was studied by using a direct binding assay. Binding of rabbit asialotransferrin, possessing a single biantenary glycan, was too weak to establish a complete binding curve, but the human asialotransferrins, possessing two glycan attachments, did yield binding data over a sufficiently wide range of concentrations for Scatchard plot analysis. At 22 degrees C, a quantity of plasma membrane equivalent to 1 mg of membrane protein bound comparable quantities (12.3-12.8 pmol) of the asialotransferrin types with an association constant of 1.5 X 10(6) M-1 for type 1, 1.4 X 10(7) M-1 for type 2, and 1.1 X 10(8) M-1 for type 3. At 4 degrees C, the number of binding sites and the association constants were reduced, more so for asialotransferrin types 2 and 3 than for type 1. At both temperatures, the shapes of the Scatchard plots for all three asialotransferrin types were similar: in the low range of bound asialoprotein (below 0.6-0.7 nM), each plot exhibited two to three convex peaks, tentatively identified as restricted domains of positive cooperativity; in the higher range, however, the plots were linear. The findings are consistent with the view that the binding sites involved in the binding of asialotransferrin are homogenous.

Animals↗
PubMed1981

Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S.

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.

Animals↗
PubMed1981

The net weight of the rabbit liver and its relevance for asialoglycoprotein clearance.

The net weight of the liver was established in 144 rabbits from isotopic determination of the quantity of residual blood in the hepatic vascular bed. Residual blood was proportional to the liver weight, amounting to 16.9% of the gross liver weight in animals that had not been exsanguinated. Net liver weights were a linear function of the body weight in the range from 0.9 kg to 5.8 kg. However, this was not true for rabbits aging 10-12 weeks where one liver out of four exhibited marked overweight due to the presence of excess quantities of metabolites. Changes in the composition of the body that accompany sexual maturation are thought to be responsible for this phenomenon. Plasma membrane function of differently sized livers was compared by determinations of the maximal hepatic clearance of a glycoprotein possessing terminal galactosyl residues (chicken alpha 1-acid glycoprotein). Results showed the clearance function to be unaffected by storage-related fluctuations in net liver weight over a two-fold range.

Animals↗
PubMed1980

Synthesis and catabolism of rabbit alpha 1-antitrypsins F and S.

The metabolic relationship between the two major forms of rabbit alpha 1-antitrypsin, F and S, was investigated by using labeling techniques in vivo and in vitro. After the injection of [14C]leucine, the S/F specific-radioactivity ratio showed characteristic changes with time: at 1 h, the ratio was high (1.2-1.4), but by later times (5-7h) it decreased to a value of approx. 1.1. Two different techniques were used to purify alpha 1-antitrypsin for labelling with iodine. The half-lives of the differentially labelled and simultaneously injected F- and S-forms were 68.1 (+/- 7.6 S.D) and 55.3 (+/- 8.1 S.D)h respectively. Combined electrophoretic and gamma-spectrometric studies provided no evidence for metabolic interconversion of the alpha 1-antitrypsin forms in the circulation. These observations suggest that rabbit alpha 1-antitrypsins F and S are, despite their close chemical composition and immunological identity, metabolically independent proteins. Therefore the possibility is raised that alpha 1-antitrypsin synthesis in rabbits is controlled by two autosomal genes or two sets of such genes.

Animals↗
PubMed1980

Multivalent interaction between asialofetuin and plasma membrane preparations from the rat liver.

Binding of bovine asialofetuin by rat liver plasma membranes was studied using different techniques for the separation of the free and bound forms of the glycoprotein and also different approaches to measure nonspecific binding. The membrane preparations had the electron microscopic appearance of a mixture of lamellae and vesicles and their lipid:protein ratios and marker enzyme profiles fell within the range of values available from the literature. The binding capacity was approximately 15 pmol of asialofetuin per milligram of membrane protein. Scatchard plots of the values obtained over a wide range of concentrations (4.8--12.6 micrograms asialofetuin per 30 micrograms membrane protein) after incubation at 22 degrees C showed pronounced nonlinearity which, in combination with evaluations according to other theoretical models, was referable to heterogeneity of binding. In sharp contrast, after incubation at 4 degrees C the Scatchard plot was linear. This difference is interpreted as the expression of a functional, rather than a chemical, heterogeneity in asialofetuin binding. The underlying mechanism is thought to be competition of galactose groups for binding sites with the result that the number of bonds varies between the galactose groups of a bound asialofetuin molecular and the hepatic lectin, depending on the concentration of the glycoprotein in the incubation mixture.

Animals↗
PubMed1980

Transferrin catabolism in mammalian species of different body sizes.

Turnover of transferrin was measured in 62 mammals from 11 species (baboon, dog, goat, guinea pig, man, mouse, pig, rabbit, rat, rhesus monkey, and sheep) using iodinelabeled transferrin of homologous and/or heterologous origin. Protein turnover was determined either from the plasma protein-bound radioactivity curves or from the slopes of total body radiation. The volume of transferrin pool, expressed as equivalent milliliters of plasma, turned over per day correlated closely with species size (r = +0.977). Using the parabolic equation, y = axb, the constants for the relationship between body weight (x) and transferrin turnover (y) were a, 22.845; and b, 0.68. However, species of comparable weights with deviating transferrin turnovers do exist within the boundaries of this relationship. From a limited number of serum transferrin estimations in all species except for rhesus, six species appeared to have transferrin concentrations in a broad middle range (2.5-3.5 mg/ml). By comparison, dog and guinea pig had markedly lower, and pig and rat had markedly higher, serum transferrin concentrations.

Animals↗
PubMed1980

Heparin inhibits thrombin binding to rabbit thoracic aorta endothelium.

Thrombin binding to freshly prepared sections of rabbit thoracic aorta was studied. After the sections had been exposed to a range of concentrations (0.1 to 3.8 IU/ml) of 125I-labeled thrombin for various periods of time at 37 degrees C, endothelial Häutchen preparations were obtained, and their radioactivity content was determined. Scatchard plot analysis of the data indicated that approximately 5.8 X 10(5) molecules of thrombin associated with each endothelial cell, with a KD of 2.6 X 10(08)M. By incubation with an excess of unlabeled thrombin, 50% of bound 125I-labeled thrombin was displaced from the endothelium in 7.3 min. Exposure of the endothelial surface to heparin (1 to 10 USP U/ml) did not significantly affect subsequent thrombin binding. However, incubation of the aorta in a thrombin solution containing 1 to 10 USP U/ml heparin did reduce enzyme binding to the endothelium by up to 60%. Similarly, the presence of heparin inhibited thrombin binding to the thoracic aorta of exsanguinated rabbits in situ. Endothelium, to which 125I-labeled thrombin was bound, lost 50% to 70% of the bound enzyme when suspended in a solution containing heparin (10 USP U/ml) and compared to the control incubated without heparin. These observations are consistent with the proposal that a major portion of endothelium-bound thrombin may be associated with pericellular heparan sulfate; heparin competes for thrombin with the heparan sulfate sites, and because of its higher affinity for thrombin, heparin displaces bound thrombin from, or inhibits binding of free thrombin by, the endothelium.

Animals↗