Modification of the biosynthesis of the protein linked carbohydrates in bovine lens epithelial cells by the eye derived growth factor and by the extracellular matrix.
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Animals↗
Biomedical subjects
E Moczar
Publications and source records attributed to E Moczar.
Structural glycoproteins from rabbit aortic media.
Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.
Animals↗
Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF). III. Control of glycoprotein and proteoglycan synthesis.
Bovine epithelial lens (BEL) cells, cultured in the presence or absence of an eye-derived growth factor (EDGF) on plastic surfaces or on extracellular matrix (ECM) laid down by corneal endothelial cells, were metabolically labelled with [3H] glucosamine and with [35S]O2+4. The newly formed glycoproteins and proteoglycans were determined and studied in the extracellular compartment. EDGF reduces radiosulfate incorporation and provides the formation of low molecular weight (LMW) proteoglycans and glycosaminoglycans. [3H]Glucosamine incorporation into the glycoprotein fractions is affected by EDGF and by ECM, but in a different manner. EDGF and ECM reduce the formation of the glycoproteins with an affinity towards the collagen, but the effect of EDGF and ECM on the other glycoproteins is different. It is suggested that the actions of EDGF and of ECM are at least partially different and mutually independent.
Animals↗
Alterations in glycopeptide and glycosaminoglycan biosynthesis in bovine lens epithelial cells cultured in the presence of an eye-derived growth factor.
D-[3H]glucosamine-labeled glycosaminoglycans and glycopeptides, obtained by Pronase digestion from bovine lens epithelial cells grown in the presence and in the absence of an eye-derived growth factor, were investigated comparatively in the extra- and pericellular compartments of 16-17th subcultures. Glycosaminoglycans and glycopeptides were separated on Ultrogel AcA 202 column and the retained glycopeptides were further separated by the successive application of affinity, ion-exchange and exclusion chromatographic methods. The following results were obtained: (1) the proportion of the label increased in hyaluronic acid and decreased in the glycopeptide fraction on stimulation with the growth factor: (2) the comparison of the chromatographic elution profiles of the glycopeptides from control and stimulated cells revealed that differences between the two cell groups were largely quantitative. An increase in the high molecular weight glycopeptides, as it was found in transformed cells, could not be demonstrated in eye-derived growth factor-treated cells.
Animals↗
Effects of retinal growth factor and of the increase of the number of subcultures on sulfated glycosaminoglycans of bovine lens epithelial cells.
Sulfated glycosaminoglycans of cultured bovine lens epithelial cells grown in the presence and in the absence of a retinal growth factor were investigated comparatively. The newly formed [35S]sulfate-labeled glycosaminoglycans were analysed in the extra-, peri- and intracellular compartments of early (4--5th) and late 17--18 h) subcultures. The following results were obtained: (1) Cultured lens epithelial cells grown in the presence or in the absence of the growth factor synthesize chondroitin 4- and 6-sulfates and dermatan sulfate, with heparan sulfate as the main component, the pericellular compartments were particularly rich in heparan sulfate; (2) The distribution pattern of the glycosaminoglycans changes during successive subcultures; the proportion of heparan sulfate increases in the pericellular compartment, the dermatan sulfate to chondroitin sulfate ratio increases in all three compartments; (3) IN contrast to the drastic decrease in the fibronectin levels in the presence of growth factor in the early subcultures, only minor differences were found between the glycosaminoglycan patterns of the treated and non-treated cells.
Animals↗
Accessibility of epsilon-amino groups of lysine to guanidination in kappa-elastin from bovine ligamentum nuchae.
Kappa-Elastin contains 3.81 lysyl residues per 1000 residues of amino acids. Among these residues, free epsilon-amino groups represent about 16 p. 100, as revealed by guanidination, the buried lysyl residues (buried epsilon-amino groups) represent about 33 p. 100, as revealed by dansylation. After drastic reduction by borohydride, no aldimine bonds were detected and only 6 p. 100 of "deeply buried" amino groups of lysyl residues were detected by a second dansylation. The remaining lysines (about 46 p. 100) are engaged or inaccessible.
Animals↗
Partial characterization of peptide fragment purified by isoelectrofocusing after organo alkaline hydrolysis of bovine ligamentum nuchae elastin.
Highly purified elastin from bovine ligamentum nuchae was submitted to partial alkaline hydrolysis (37 degree C, 72 H, 1 N KOH in 80 p. 100 aqueous ethanol). The non-coacervable fractions were submitted to isoelectrofocusing and five kappa-elastin fractions were obtained. The amino-acid compositions, the N-terminal amino-acids, the molecular weights and the thermolysin digests of each fraction were determined by various techniques. The average MW was about 14,500 (150 - 166 amino-acids). These results suggest that the distribution of cross-linking agents in fibrous elastin may not be uniform. The results also show that in certain cross-linked regions of similar molecular weight and size appearing to be composed of different polypeptides sequences containing different amounts of cross-links.
Animals↗
Differentiation of the polysaccharide side-chains of glycoproteins by a fingerprinting technique: heterogeneity of human gastric mucin.
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ABO Blood-Group System↗
Glycosaminoglycans in elastase induced emphysema.
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Animals↗
Biodegradable arterial prosthesis from rat aorta.
The preparation and potential clinical use of biodegradable microarterial grafts from rat aorta were investigated. Trypsin treated arterial segments were coated with heparin or chondroitin sulfate to reduce thrombogenicity. The samples were crosslinked with formaldehyde vapors at 4 degrees C. 50 - 100 microgram glycosaminoglycans taken up per mg aorta dry weight were resistant to washing with water for 24 hrs. The covalent crosslinks introduced by formaldehyde and resistance of the grafts to proteolytic degradation. The treated grafts were implanted on 70 rats in an infrarenal aortic position. The permeability of the aldehyde crosslinked prosthesis after 21 days by patency test was lower than the patency ratio measured with fresh autologous grafts. The glycosaminoglycans associated with the prosthesis improve the patency of the crosslinked grafts by about 48%. The resistance to bacterial collagenase of the excised grafts decreased with progressing time of implantation. In the permeable prosthesis and in the contiguous aorta, elastolytic activity was demonstrated by radial diffusion in elastin-agar gels. The grafts removed after 21 days of implantation were surrounded with scar tissue. In contrast to fresh aorta, the macromolecular hydroxyprolin in the scar was readily solubilized with pepsin. The presence of the fragmented elastin and collagen fibers in the excised graft is in favour of their resorption "in vivo".
Animals↗
Biochemical and pharmacological properties of a neurotoxic protein isolated from the blood serum of heavily burned patients.
Blood serum of heavily burned patients contains neurotoxic substances which are not present in normal control sera. In the present paper, we describe the purification by gel filtration and ultracentrifugation of such a neurotoxic factor. The purified factor appeared to be a high molecular weight (2 to 3.10(6) daltons) lipoprotein. This factor was present in all the sera of patients with more than 35 per cent of the body surface burned. When injected into rabbits the lipoprotein caused a flattening of the EEG tracing, then trembling and convulsions with bursts of spikes on the EEG. The activity of this neurotoxic substance was enhanced when the permeability of the blood-brain barrier was increased by previous intraventricular injection of collagenase. The presence of such a neurotoxic factor in the blood serum of burned patients, together with the increased serum collagenase activity they exhibit may explain the neurotoxic symptoms observed in them.
Animals↗
Biochemical and pharmacological properties of a cardiotoxic factor isolated from the blood serum of burned patients.
Blood serum of severely burned patients contains several substances which are not present in normal sera. One of these substances, a small protein of an approximate molecular weight of 12 to 14,000 daltons displayed a toxic action on the circulatory system. This cardiotoxic factor was obtained in a purified form by alcohol precipitation followed by gel filtration and carboxymethyl cellulose chromatography. The purified preparation seems to be a low molecular weight protein of about 8000 daltons. The biological effects of this substance consist essentially of alterations of the ECG pattern, indicating decreased cardiac output and ischaemia of the cardial muscle. The blood pressure decreases and respiratory function is also altered. The presence of such a toxic factor in sufficient concentration in the blood serum may explain the cardiovascular complications observed in some burned patients.
Animals↗
Chemical modification of kappa-elastin from bovine ligamentum nuchae.
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Amino Acids↗
The specificity of sialytransferases using glycosylated lysozyme derivatives as substrates.
Galactose, lactose, N-acetylgalactosamine, N-acetylglucosamine and fibrinoglycopeptides were bound to lysozyme by different linkages. These glycosylated lysozymes were tested as N-acetylneuraminic acid acceptors using particular sialytransferase preparations from frog and bovine liver and from bovine and porcine submandibular glands. Desialylated fetuin served as reference compound. Galactose residues of desialo-fetuin and lysozyme-lactose are sialylated by all four sialytransferases tested, galactose bound to lysozyme via a phenylazo group is inactive with the enzyme from bovine submandibular gland, and galactose bound directly to lysozyme serves as substrate only for the frog liver sialytransferase. Lysozyme-phenylazo-N-acetylgalactosamine is active only with the sialytransferase from bovine sumbandibular gland. N-Acetylglucosamine derivatives of lysozyme are inactive with all sialytransferases tested. These observations are discussed in the light of the natural substrates for the sialytransferases investigated.
Glycopeptides↗
Peptides obtained from elastin by hydrolysis with aqueous ethanolic potassium hydroxide.
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Alanine↗
Guanidination of kappa-elastin from bovine ligamentum nuchae.
Kappa-Elastin is exhaustively guanidinated in order to chemically modify the pre-existing lysine content to homoarginine. However the pre-existing lysine content is only modified to homoarginine content about 50 p.cent. The other lysyl-bonds are buried or/and engaged to aldi-imine bonds or other bonds. Therefore the first guanidinated kappa-Elastin is submitted to partial acide hydrolysis (HCL, 1 N, 110 degrees C for 2 hours) and submitted to a second exhaustive guanidination. The pre-existing lysin content in kappa-Elastin almost quantitively disappeared and is transformed to the corresponding homoarginine. These results are similar to those obtained by photolysis of (Iso) desmosine residues by UV light which will liberate "new" lysine containing peptides from kappa-Elastin.
Amino Acids↗
Two-dimensional separation of glycopeptides and charged oligosaccharides on silica thin layers.
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Chromatography, Thin Layer↗
Glycoproteins from the aorta.
This review deals with the data on isolation techniques, structure, physicochemical properties and biological function of glycoproteins isolated from the aorta. Some glycoproteins extracted at low ionic strengths (soluble glycoproteins) are immunologically distinct from serum glycoproteins. The designation "structural glycoprotein" was proposed for glycoproteins associated with the collagen elastin matrix. These glycoproteins are solubilized by dissociative and reducing buffers. A selective hydrolysis of the insoluble collagen prior to their extraction was also reported. The molecular weights are in the range of 53 - 72.000 and 35.000 - 27.000 daltons for the soluble and the structural glycoproteins respectively. The aorta glycoproteins are rich in polar aminoacids. The primary structure of glycoproteins from aorta is not yet established. The electrophoretic mobility and sugar content of some glycoproteins extracted at low ionic forces change with the maturation and sex. The biosynthesis of a soluble glycoprotein (glycoprotein B) and of structural glycoproteins by aortic smooth muscle cells was demonstrated "in vitro". The structural glycoproteins appear as microfibrillae in electron microscopy. Their self-aggregation may be due to hydrophobic interactions. The association of microfibrillar glycoprotein with elastin in connective tissues and in the culture medium of aortic smooth muscle cells suggests the role of glycoprotein-elastin interactions in morphogenesis.
Amino Acids↗