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E Regoeczi

Publications and source records attributed to E Regoeczi.

At least 37 records · Page 2Linked to original sources
PubMed1988

Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A.

The content and distribution of transferrin receptors (Tf-R) in suspended adult rat hepatocytes were studied using 125I-protein A in combination with either a monoclonal (MRC OX-26) or a polyclonal antibody to Tf-R. Internal receptors were made accessible by permeabilization with digitonin. The number of Tf-R detected depended on the batch of collagenase used for liver perfusion. By using the monoclonal reagent in conjunction with the less damaging of two batches of the enzyme, 129,000 receptors were found per cell, with 47,000 (37%) of these on the surface. The polyclonal reagent yielded Tf-R numbers which were consistently higher than those obtained with MRC OX-26. This difference is interpreted as being due to the binding of several (on the average 5-6) molecules of polyclonal IgG per molecule of Tf-R. Remarkably, transferrin binding by Tf-R was not affected by this cluster of associated IgG and the overlayer of protein A. Parallel studies with 131I-transferrin in a simplified binding assay system yielded surface Tf-R estimates which, in most cases, were close to the values obtained with MRC OX-26. After prolonged exposure to collagenase, the ligand-binding capacity of Tf-R was more affected than its immunoreactivity. In preliminary studies, monensin (10 microM) produced a 32%-50% shift of Tf-R from the surface to the inside, whereas short-term incubation with epidermal growth factor (0.17 mM) brought about no clear-cut Tf-R redistribution.

Animals↗
PubMed1987

Metabolic stability of the fucose in rat transferrin.

The metabolic behaviour of the chitobiose core fucose that is a natural constituent of a large proportion of rat transferrin molecules was studied in rats comparatively to that of the polypeptide portion of the glycoprotein by using appropriate labels ([3H]fucose and 125I) and affinity chromatographic techniques (lentil-Sepharose). No evidence was obtained to suggest that this residue is cleaved from the glycan in significant amounts before removal of the entire glycoprotein for catabolism. Similarly, [14C]fucose linked to GlcNAc residues in the antennae of human asialotransferrin was being eliminated in pigeons at the same rate as the polypeptide itself. It is concluded that in spite of transferrin's exposure to the cellular milieu, the fate of its fucose is distinctly different from that of the same in plasma membrane glycoproteins.

Animals↗
PubMed1987

The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin.

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.

Animals↗
PubMed1987

Distribution of antithrombin III in rabbits: role of host and protein.

Unlike in the case of some other species, the plasma curve of iodine-labeled antithrombin III (I-AT-III) in rabbits requires fitting with a three-term exponential function for obtaining reliable estimates of the catabolic rate and distribution of I-AT-III among various body compartments (Carlson, Atencio, and Simon. J. Clin. Invest. 74: 191-199, 1984). To decide whether this phenomenon is referable to the host or the protein, the behavior of rabbit and human I-AT-III was comparatively analyzed in rabbits. Data obtained with rabbit I-AT-III confirmed the findings by Carlson and co-workers. Human I-AT-III assumed a distribution that closely paralleled that of homologous I-AT-III, thus suggesting that the pattern of distribution is determined by the host species rather than its AT-III. Rabbits metabolized human I-AT-III 1.61 times faster than homologous I-AT-III by an unknown mechanism not involving immune response; a facet that may prove useful for the identification of the sites of catabolism of AT-III. The exponent of the body weight was calculated for the relationship between species size and AT-III turnover. A value of 0.5 was obtained that is distinctly lower than the exponents found earlier for some other plasma proteins.

Animals↗
PubMed1987

Why do total-body decay curves of iodine-labeled proteins begin with a delay?

The initial delay that occurs in total-body radiation curves reaching their single-exponential slopes was analyzed from 106 experiments involving several mammalian species (guinea pig, mouse, rabbit, and rat) and plasma proteins (alpha 1-acid glycoprotein, antithrombin III, fibrinogen, immunoglobulin G, and transferrin) in 14 different combinations. The time interval (Td) between injection and the intercept of the slope with the full-dose value was adopted as a measure of curve nonideality. The overall mean Td was 6.6 h, but individual values showed a significant correlation to protein half-lives, whereby proteins of unequal metabolic properties exhibited different mean Td values. Targeting protein to the liver abolished delay. Choice of the isotope (125I or 131I) and size of the labeled protein had no influence on the magnitude of delay. Whole-body radiation curves of animals that received [125I]iodotyrosines, Na131I, or 131I-polyvinylpyrrolidone exhibited no initial delays. These results do not support the earlier notion that delay is caused by a redistribution of the labeled protein in the body to radiometrically more favorable sites. However, they are compatible with the assumption that delayed passage of a protein dose through the extracellular matrix and/or retarded transfer of proteolytic products from extravascular catabolic sites to plasma may be responsible for the phenomenon.

Animals↗
PubMed1986

Preferential hepatic uptake of iron from rat asialotransferrin: possible engagement of two receptors.

Hepatic iron uptake from and degradation of rat asialotransferrin prepared from the least anionic (major) component of rat transferrin were studied in intact rats. In experiments lasting 60-90 min, rat asialotransferrin delivered a three to four times larger fraction of the Fe dose to the liver than rat transferrin. Variations in the concentration of endogenous circulating rat 2Fe-transferrin by up to 300% failed to affect the enhanced hepatic delivery of Fe from rat asialotransferrin. However, pretreating the animals with a large dose of asialomucin, or fully sialylated human transferrin, or a combination of both did affect the delivery. In all cases, rat asialotransferrin delivered Fe to the liver at rates comparable with those seen with rat transferrin. The reason for the efficacy of human transferrin was clarified in competitive binding studies on rat hepatocytes and reticulocytes, which showed that human transferrin possessed an approximately sevenfold higher affinity for rat transferrin receptors than the homologous protein. These findings suggest that the enhanced hepatic uptake of Fe from rat asialotransferrin is mediated by simultaneous binding of the ligand both through its glycan and transferrin receptor affinity site. Pretreatment with asialomucin and human transferrin had no suppressing effect on basal hepatic delivery of iron from rat 2Fe-transferrin. The data suggest that deposition of a significant fraction of Fe in rat liver from rat transferrin is likely to take place by a mechanism not involving transferrin receptors. Desialylation shortened the metabolic half-life of rat transferrin from 33 to 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Alpha-1-acute phase globulin in the blood of tunicamycin-injected rats. Isolation of the non-glycosylated form, its inhibitory properties and synthesis in liver slices.

Plasma of rats injected with tunicamycin (2 micrograms/g body wt) shows on crossed immunoelectrophoresis the presence of an additional, slowly migrating component of alpha 1-acute-phase globulin (alpha 1-AP-globulin). The native and modified forms of alpha 1-AP-globulin were jointly isolated on the column of immobilized antibodies and then separated by chromatofocusing on polybuffer exchanger. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate demonstrated that tunicamycin-induced form of rat alpha 1-AP-globulin has Mr of 50,000 and is devoid of carbohydrates as inferred from the lack of staining with Schiff reagent. However, during incubation with papain in vitro it is only slightly less effective than mature glycosylated alpha 1-AP-globulin (Mr 68 000) in inhibiting hydrolysis of CBZ-Lys-ONp. Incubation of liver slices from control and tunicamycin-injected rats with 14C-leucine demonstrated that tunicamycin reduces synthesis and release to the medium of alpha 1-AP-globulin and some other plasma proteins, but the proportion of aglyco-alpha 1-AP-globulin is higher than in plasma.

Acute-Phase Proteins↗
PubMed1985

A simple bioassay for monocyte-derived hepatocyte stimulating factor: increased synthesis of alpha 2-macroglobulin and reduced synthesis of albumin by cultured rat hepatocytes.

Cytokines released from monocytes upon stimulation by lipopolysaccharide cause a number of cells to undergo proliferative and synthetic changes. At least one of these cytokines affects hepatocytes in vivo causing increased synthesis of a series of acute-phase proteins. We have established an in vitro micro-assay for hepatocyte stimulating factor (HSF) using primary cultures of normal rat hepatocytes. Measurement of increased synthesis of alpha 2-macroglobulin and decreased synthesis of albumin caused by exogenously added factor constitute a sensitive parameter for quantifying HSF. For comparing various cytokines preparations, we have defined a unit of HSF activity in terms of a stimulation index. We have used this assay to follow some preliminary attempts to isolate the factors responsible for stimulation of synthesis of acute-phase reactant by the liver.

Acute-Phase Proteins↗
PubMed1985

The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat.

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.

Alkaloids↗
PubMed1985

Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors.

The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site.

Animals↗
PubMed1985

Lactoferrin catabolism in the rat liver.

The hepatic uptake and degradation of human diferric 125I-lactoferrin by the liver of the intact rat were studied. After intravenous injection, the tracer was rapidly cleared by the liver, probably by adsorptive pinocytosis, as inferred from observations with a 3,470-fold dose range. Endocytosed lactoferrin was transferred, with a delay, from a light-density subcellular particle to an organelle that had a density similar to lysosomes. The loss of protein bound 125I from the liver was very slow (half-life 2.7 h), and its rate matched closely that of human asialotransferrin type 3. Lactoferrin was found to be a poor substrate for lysosomal hydrolases in vitro. Fucoidin effected the release of a portion of lactoferrin from the liver back into the plasma. By using this agent, indirect evidence was obtained suggesting that a fraction of lactoferrin is being repeatedly endo- and exocytosed (diacytosed) by the liver over prolonged periods of time. Fucosylation failed to impart lactoferrinlike properties on human asialotransferrin type 1, although the derivatized protein exhibited a less than or equal to 10-fold increase in affinity for the liver relative to the parent molecule.

Animals↗
PubMed1985

Interaction of human lactoferrin with the rat liver.

Binding of human lactoferrin (hLf) by purified rat liver plasma membranes was studied to clarify whether the liver possesses specific hLf receptors. The binding was rapid between 4 degrees and 37 degrees C, with a pH optimum close to 5.0. At 22 degrees C and in glycine-NaOH (5 mM, pH 7.4) containing 150 mM NaCl and 0.5% albumin, 1 microgram of membrane bound a maximum of 11.8 ng hLf. The dissociation constant of the interaction was 1.6 X 10(-7) M. Other proteins of high isoelectric points (lactoperoxidase, lysozyme, and particularly salmine sulfate) and a piperazine derivative inhibited hLf binding in a concentration-dependent manner. In contrast, monosaccharides (galactose, N-acetylgalactosamine, mannose, and fucose) were ineffective. By omitting NaCl from the incubation buffer, binding was increased 3.6-fold. Erythrocyte ghosts bound hLf less firmly and alveolar macrophages more firmly than hepatic plasma membranes. Liver cell fractionations performed after the intravenous injection of labeled hLf showed that approximately 88% of the hepatic radioligand was associated with parenchymal cells. When binding was expressed per unit of cell volume, however, more hLf was present in nonparenchymal than in parenchymal cells, implying that the above value was determined by the relative cell masses rather than affinities alone. It is concluded that the binding of hLf by hepatic plasma membranes is electrostatic, i.e., is mediated by the cationic nature of the ligand, and that it is explicable in terms of a "specific nonreceptor interaction" of the generalized type proposed by Cuatrecasas and Hollenberg (Adv. Protein Chem. 30: 251-451, 1976).

Animals↗
PubMed1984

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.

Acute-Phase Proteins↗
PubMed1984

Two populations of prelysosomal structures transporting asialoglycoproteins in rat liver.

Analyses by differential centrifugation of liver homogenates from rats that had received 131I-labeled asialoorosomucoid showed that, 1 min after injection, most of the intracellular ligand was associated with a particle that did not sediment at 2.5 X 10(5) g-min. However, by 10 min, undigested ligand became associated with a particle that did sediment at this speed. On analytical ultracentrifugation in sucrose gradients, both kinds of particles exhibited low densities (1.11-1.13 g X ml-1). In contrast to asialoorosomucoid, 125I-labeled asialotransferrin type 3, under noncatabolic conditions, remained largely confined to the nonsedimenting particle regardless of the duration of the study. Induction of catabolism of asialotransferrin was accompanied by the appearance of the ligand in the sedimentable particle. The nonsedimentable particle was separated by immunoadsorption from other subcellular particles contained in the low-density subcellular fraction. The adsorbant , prepared by immobilizing purified antibodies to the Gal/GalN-specific lectin from rat liver on coated polyacrylamide beads, removed 75-80% of the asialoorosomucoid and transferrin binding capacities present, together with a similar portion of the radioligands tested (asialoorosomucoid, asialotransferrin type 3, and human diferric transferrin). Significantly, the sialytransferase activity remained unadsorbed. From these findings, the nonsedimentable particle appears to be involved in the transport of ligands destined to such diverse fates as exocytosis or lysosomal degradation. The sedimentable particle, on the other hand, seems to represent a link between the first particle and the lysosome.

Animals↗
PubMed1984

Partial resialylation of human asialotransferrin types 1 and 2 in the rat.

125I-labeled asialotransferrin types 1 and 2 were administered in small doses to rats. The protein still in the plasma after 1-12 h was partially repurified and electrophoresed at pH 8.1, together with a transferrin standard that is composed of all six forms of the protein with respect to sialic acid content. The electrophoretic mobility of both asialotransferrins increased with time, type 2 being affected sooner than type 1. The changed mobility was due to increased electronegativity that was fully reversible by treatment of the samples with neuraminidase, thus identifying the underlying cause as partial resialylation. Asialotransferrin incubated in vitro with serum, plasma, or whole blood for 16 h exhibited no change in electrophoretic mobility. In conjunction with an earlier study on asialotransferrin type 3, it was found that the apparent speeds of resialylation of the three asialotransferrins were in the same order as their affinities for the asialoglycoprotein-binding hepatic lectin. This suggests the involvement of an endo- rather than of an ecto-transferase. Transfer of 59Fe from asialotransferrins to the liver was used to monitor the frequency of hepatocyte-asialotransferrin interactions. Iron deposition in the liver took place much more rapidly than the appearance of detectable quantities of partially resialylated asialotransferrin molecules in the circulation. It is concluded that each asialotransferrin molecule probably undergoes several passages through the hepatocyte before its glycans become modified.

Animals↗
PubMed1984

Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver.

Rats were given an intravenous dose (1-2 micrograms/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10-20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.

Animals↗