Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10(7) Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true 'mucin', the large size and many other properties are quite 'mucin-like'. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl/GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.
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PMID:High molecular weight mucin-like glycoproteins of the bovine interphotoreceptor matrix. 154 29

We isolated mucin-like glycoproteins from the conditioned medium of primary hamster tracheal epithelial (HTE) cell culture and characterized them biochemically and immunologically. These glycoproteins were purified on Sepharose CL-4B after Streptomyces hyaluronidase treatment and then by CsCl-density-gradient centrifugation in the presence of 4 M-guanidinium chloride. The purified glycoproteins were resistant to digestion by chondroitin AC lyase, heparinase, heparitinase and endo-N-acetylglucosaminidases A, D and H, but susceptible to endo-beta-galactosidase and keratanase. SDS/PAGE demonstrated no contamination by low-molecular-mass proteins. The purified glycoproteins showed a peak buoyant density of 1.56 g/ml in CsCl-density-gradient centrifugation, and contained 10% peptide and 90% carbohydrate by weight. Carbohydrates in these glycoproteins contained N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, sialic acid and a trace amount of mannose, but no uronic acid. Serine and threonine together accounted for 27% of the total amino acid residues. In addition, the mucin-like glycoproteins exhibited blood-group A and B activities, and very strong inhibitory activity for influenza A virus haemagglutination. With the use of the purified glycoprotein as an antigen, six monoclonal antibodies that stained mucus granules in hamster tracheal epithelium were obtained. We characterized the antibody produced by one of the clones, HM D46. We conclude that HTE cells cultured in the serum-free medium secrete a glycoprotein with physicochemical properties similar to those known in various airways mucins.
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PMID:Mucin-like glycoprotein secreted by cultured hamster tracheal epithelial cells. Biochemical and immunological characterization. 165

The synthesis and secretion of mucin-like high-molecular glycoprotein was studied in 2 human colon cancer cell lines that spontaneously differentiate in culture (Caco-2 and T84) and in 2 cell lines that do not spontaneously differentiate (LS174T and HT29). Mucin, quantitated by 3H-glucosamine labelling and chromatography on Sepharose CL-4B was found to be produced by all 4 cell lines. The mucinous nature of the labelled high-molecular glycoprotein was verified by enzymatic degradation treatments (heparinase, hyaluronidase, chondroitinase ABC, and N-glycanase), alkaline-borohydride treatment, inhibition of labelling by the glycosylation inhibitor benzyl-alpha-GalNAc, and by CsCl-density-gradient centrifugation. In all 4 cell lines, an inverse correlation of mucin synthesis with cell density was demonstrated. In Caco-2 cells, the spontaneous post-confluent enterocytic differentiation with increased brush-border enzyme expression was associated with a decrease in mucin synthesis and in the activities of polypeptidyl GalNAc transferase and beta 1,3-galactosyltransferase activity. Using cDNA probes for 2 distinct human intestinal mucins (MUC2 and MUC3), we found that all 4 colon cancer cell lines expressed mucin message, but the types of mucin mRNA expressed differed. These data indicate that mucin-like glycoproteins can be synthesized by cell lines derived from non-mucinous colon cancer, whether or not they undergo spontaneous differentiation in culture. These cell lines may serve as in vitro models for studying apomucin heterogeneity and control of mucin gene expression.
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PMID:Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. 172 5

To clarify the role of electrostatic interactions in the binding of Sindbis virus (SNV) to cell membrane receptors, we investigated the effect of different polyions on the initial steps of infection of Vero cells by the virus. Several polyanions (mucin, heparin, polygalacturonic acid) and polycations (polylysine, protamine, polybrene) were able to reduce the replication of SNV when present in the viral adsorption period, whereas others (chondroitin sulfate, polymyxin B sulfate, histone) were devoid of any activity. Therefore the electric charge alone is not sufficient to explain the action of compounds. The effects of polyions on receptor binding, on bound virus, and on internalized virus have been examined. All the drugs inhibited SNV infection by affecting its binding to the cellular receptor. The results indicated that heparin and mucin act directly on the virus particle while polycations bind to the cell membrane receptor for the virus, protamine being effective on both targets. Since among polyanions glycosaminoglycans showed a strong inhibiting activity, the involvement of these molecules in the virus surface receptor was assessed by enzyme digestion of cell membrane with heparinase and chondroitin ABC lyase.
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PMID:Effect of polyions on the early events of Sindbis virus infection of Vero cells. 175 5

Organ culture of guinea pig trachea was performed in the presence of [35S]sulfate in order to characterize the sulfated glycoproteins released from the respiratory epithelium and mucosa. The sulfated macromolecules that were synthesized during a 6-h incorporation were separated by CsBr density-gradient centrifugation and gel-filtration chromatography successively. Most of the sulfated secreted macromolecules corresponded to a population of glycoproteins sensitive to reductive beta-elimination but resistant to both chondroitinase ABC and heparinase. These glycoproteins had different buoyant densities (ranging from 1.48 g/ml to 1.16 g/ml) and could be subfractionated according to molecular mass. A major part of the radioactivity was incorporated into high-molecular-mass mucins that were excluded from a Sepharose CL-2B column and did not penetrate into polyacrylamide gel in PAGE. However, a mixture of sulfated O-glycoproteins of much lower molecular mass was also characterized in addition to low amounts of chondroitin sulfate. Epithelial goblet cells are the predominant mucin-containing cells of the respiratory guinea pig trachea. Our results suggest that a wide range of sulfated O-glycoproteins are secreted by the guinea pig tracheal mucosa.
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PMID:Sulfated O-glycoproteins secreted by guinea pig trachea in organ culture. 189 37

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.
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PMID:An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates. 262 58

The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by cystic fibrosis cells (CFPAC-1), a pancreatic cancer cell line derived from a patient with cystic fibrosis, and pancreatic cancer (SW-1990) cell lines. High molecular weight glycoproteins (HMG) were quantified by [3H]-glucosamine labeling and chromatography on sepharose CL-4B. Mucin gene expression was determined by using cDNA probes for 2 distinct intestinal mucins (MUC2 and MUC3) and one stomach mucin (MUC1). The specific mucin core epitopes were confirmed by immunoblots using antibodies that recognize T, Tn, sialosyl Tn, MUC1, MUC2, and MUC3. The results of these experiments demonstrate that CFPAC-1 cells contained 1.25 fold and 1.4 fold more HMG in the membrane and cytosolic fractions, however, secreted 4-fold more HMG into the medium compared to SW-1990 cells. The HMG of SW-1990 was found to be mucinous in nature and not proteoglycans, as it was not susceptible to hyalurinidase, heparinase and chondroitinase ABC. The HMG of CFPAC-1 was also predominantly (80%) mucinous but with small amounts of proteoglycans. mRNA and immunoblot analysis suggest that these CFPAC-1 and SW-1990 cells predominantly express MUC1 apomucin, small amounts of MUC2 apomucin, and no MUC3. Pulse chase labeling and immunoprecipitation of MUC1 type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin gene product were present in both cell lines, corresponding to the known length polymorphism of this mucin. Both T and Tn antigens were significantly higher in CFPAC-1 and SW-1990 cells as compared to sialosyl Tn antigen. These findings were associated with the increased activities of polypeptidyl N-acetylgalactosaminyl transferase and b1,3-galactosyltransferase. These investigations demonstrate for the first time that cystic fibrosis cells (CFPAC-1) secrete and synthesize high amounts of mucin which is associated with high levels of MUC1 mRNA, low levels of MUC2 mRNA and non detectable MUC3 mRNA.
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PMID:Cystic fibrosis and pancreatic cancer cells synthesize and secrete MUC1 type mucin gene product. 754 50

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
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PMID:Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin. 787 96

A human endometrial adenocarcinoma cell line (Ishikawa) has been shown to incorporate [3H]glucosamine and to secrete a radiolabeled high molecular weight compound which is excluded from a Sepharose CL-2B column. The excluded material was resistant to hyaluronidase, chondroitinase ABC, and heparinase. These findings rule out the possibility of this material being a proteoglycan. The susceptibility of this material to digestion with pronase, neuraminidase, and alkaline borohydride treatment strongly suggests that the excluded material is an O-glycosidic glycoprotein. The glycoprotein secreted by Ishikawa cells (ICGP) did not react immunologically with antibodies against either lactoferrin or fibronectin, but did react with an antibody made against tracheal mucin. Conversely, immunoblot analysis revealed that an antibody made against ICGP did not recognize hyaluronic acid, chondroitin, heparin, nasal turbinate mucin, bovine submaxillary gland mucin, lactoferrin, or fibronectin, but did recognize tracheal mucin. Analysis of ICGP amino acid and carbohydrate composition showed that it is rich in serine, threonine, glutamic acid, aspartic acid, and N-acetylneuraminic acid. In this respect, ICGP differs from other mucins, even though it is immunologically similar to respiratory mucin; hence we may consider ICGP to be a mucin-like glycoprotein. Secretion of ICGP can be modulated by Ca(2+)-ionophore and other mucus secretagogues, such as platelet activating factor, carbachol, and monocyte/macrophage mucus secretagogue, all mediators of lung inflammation. Ishikawa cells and anti-ICGP antibody may be used in studies on in vitro regulation of mucin-like glycoprotein synthesis and secretion in the respiratory tract as well as in the endometrium.
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PMID:Characterization of a unique mucin-like glycoprotein secreted by a human endometrial adenocarcinoma cell line (Ishikawa). 818 54

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mucinlike glycoproteins from cat tracheal gland cells in primary culture. 821 86


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