Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A low-molecular-weight, monomeric, mucin-type glycoprotein (MG2) has been isolated from human submandibular-sublingual saliva. Initial purification involved sequential gel-filtration on Sephadex G-200 and Sepharose CL-2B, the latter in the presence of 6M urea. Fractions containing MG2 were next separated from contaminating secretory IgA by immunoaffinity chromatography or recycling through Sephadex G-200. Mucin fractions were 14C-labeled by reductive methylation, and then the final purification-step entailed recycling radiolabeled materials through Sephadex G-200. Radiolabeling aided in the assessment of purity, as judged by SDS-PAGE and ion-exchange chromatography. The carbohydrate portion accounted for 69.6% of the recovered weight and was composed of N-acetyl-glucosamine, N-acetylgalactosamine, galactose, fucose, and N-acetylneuraminic acid. Sulfate was also present. The protein comprised 30.4% of the recovered weight with threonine, serine, proline, and glycine accounting for 75.2% of the total amino acids. The oligosaccharides were alkali-labile, indicating an O-glycosyl linkage to the peptide. The mucin was weakly acidic and had an estimated mol. wt. of 200 000-250 000.
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PMID:Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva. 713 59

We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.
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PMID:Regulated and unregulated pathways for MUC2 mucin secretion in human colonic LS180 adenocarcinoma cells are distinct. 749 1

Previous studies have shown that Schistosoma mansoni excretory-secretory polypeptides (ESP) inhibit various internal defense functions of hemocytes from Biomphalaria glabrata and that plasma also may exert a modulatory effect on hemocyte activity. To better understand how plasma may influence hemocyte-schistosome interactions in inbred strains of snails, biotinylated ESP (b-ESP) were used as probes to identify ESP-reactive plasma components and partially characterize the nature of their binding interactions. In a plasma binding assay, b-ESP bound in a dose-dependent fashion to immobilized snail plasma, although no quantitative differences in ESP reactivity to plasma of schistosome-susceptible (M-line) and -resistant (13-16-R1) snail strains were detected. Moreover, co-incubation of b-ESP with homologous plasma or pretreatment of plasma with nonbiotinylated ESP in the plate assay significantly reduced b-ESP binding to immobilized plasma, indicating a specific interaction between ESP and plasma. Pretreatment of b-ESP with mannose, porcine stomach mucin, fetuin, and asialofetuin resulted in a significant inhibition of b-ESP binding to plasma, whereas pretreatment of immobilized plasma with a cocktail of monosaccharides (including mannose), porcine stomach mucin, and fetuin had no inhibitory effect. These data suggest the presence of a carbohydrate-binding protein(s) in sporocyst ESP that is targeted to plasma glycoconjugates. Based on the carbohydrate content of the major inhibitory glycoproteins, galactosyl and n-acetyl-galactosaminyl sugars may represent putative determinants for the lectin-like ESP molecule(s). However, ESP binding to plasma from M-line and 13-16-R1 B. glabrata strains exhibited similar sugar and glycoprotein inhibition patterns. SDS-PAGE and electroblot analyses further demonstrated that ESP bound to a subset of separated plasma polypeptides, most prominently to a doublet of 52 and 54 kDa, and a complex of proteins with molecular masses greater than 150 kDa. Other polypeptides exhibiting weaker binding interactions included components of 34, 60, 64, 86, and 125 kDa. Although both M-line and 13-16-R1 snail strains contained a similar complement of ESP-binding plasma proteins, ESP binding to the 34- and 86-kDa proteins occurred at higher frequencies in susceptible M-line plasma. It is concluded that ESP, released during in vitro transformation of S. mansoni miracidia, contain carbohydrate-reactive proteins capable of selectively binding to components of snail plasma. Quantitative differences between snail strains in the occurrence of ESP-binding plasma molecules was documented.
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PMID:Schistosoma mansoni: excretory-secretory polypeptides exhibit selective binding to plasma components of the snail Biomphalaria glabrata. 749 26

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.
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PMID:Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization. 752 92

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.
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PMID:Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant. 753 64

The probably sole constituent of the filamentous brush border glycocalyx, which has been defined on the basis of electron microscopic data as a set of filaments radiating from the tip of rabbit intestinal brush border microvilli, has been purified. It consists of a mucin-type glycoprotein that can be solubilized by either Triton extraction or papain treatment of the brush border membrane vesicles but is insensitive to phosphatidylinositol phospholipase C. The detergent- and papain-solubilized forms both have the same apparent molecular mass of 400 kDa (SDS/PAGE). This suggests that the filamentous brush border glycocalyx may be anchored to the membrane by a small hydrophobic peptidic tail. Ser, Thr, Pro and Ala amount to 65% of the protein core amino acid residues. The glycosidic moiety, which amounts to 73% of the molecular mass, has high O-acetylated sialic acid contents. A monoclonal antibody (3A4) raised against the purified material was produced which specifically recognized the 400-kDa band by immunoprecipitation and immunoblotting, and the filamentous brush border glycocalyx of villus enterocytes when jejunum sections were immunolabelled. The 3A4 determinant was identified with a filamentous brush border glycocalyx-specific carbohydrate structure containing an O-acetylated sialic acid. The fact that the labeled glycocalyx was anchored entirely in a membrane microdomain at the tip of the microvilli shows that mature enterocytes are hyper-polarized epithelial cells.
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PMID:The filamentous brush border glycocalyx, a mucin-like marker of enterocyte hyper-polarization. 753 95

This paper describes a novel technique for purifying glycoproteins from porcine gastric mucus by preparative polyacrylamide gel electrophoresis and electroelution. The method is based on the observation that the high-molecular-weight buffer/SDS-soluble mucins do not penetrate through the polyacrylamide gel, but remain on the gel surface. Mucus solution extracted with 6 M urea was fractionated on Sepharose CL-2B column and Vo peak mucin was submitted to purification by preparative polyacrylamide gel electrophoresis (22 h). Nonpenetrated mucin layer was electroeluted from the gel after the reversing of electrode polarity (3 h). A comparison of mucin preparations purified by our method and by CsCl density gradient centrifugation indicated that the GalNAc/protein and GalNAc/DNA ratios were three times higher than those of the first method. The method is a relatively short and efficient procedure and yields pure mucin preparation free of contaminating proteins and nucleic acids.
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PMID:The use of preparative polyacrylamide gel electrophoresis and electroelution for purification of mucus glycoproteins. 754 Aug 8

Four new monocot lectins from the tubers of araceous plants, namely, Arisaema consanguineum Schott (ACA), A. curvatum Kunth (ACmA) and Sauromatum guttatum Schott (SGA) from the tribe Areae, and Gonatanthus pumilus D. Don (GPA) from the tribe Colocasieae have been purified by affinity chromatography on asialofetuin-linked amino activated silica beads. These lectins possess similar physicochemical and biological properties. All the lectins gave a single peak on HPLC size exclusion and cation exchange columns, and a single band on PAGE, (pH 4.5). In SDS-PAGE, all the lectins gave a single band corresponding to a subunit of M(r) 1,3000. All the lectins yielded multiple peaks on anion-exchange column, multiple bands on non-denatured PAGE (pH 8.3) and a family of bands on isoelectric focusing. The lectins agglutinate rabbit, rat and sheep red blood cells (RBCs) but are inactive towards human ABO erythrocytes. The haemagglutination activity of these lectins is inhibited by asialofetuin only, while simple sugars/derivatives including chitin, porcine mucin and fetuin did not react. In serological studies against rabbit anti-SGA serum, all four lectins produced immunoprecipitin lines. The lectins within each tribe were identical but the lectins belonging to the tribe Areae were only partially identical to the lectins from the tribe Colocasieae.
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PMID:Purification and properties of four monocot lectins from the family Araceae. 754 57

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.
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PMID:Biosynthesis of rat MUC2 in colon and its analogy with human MUC2. 761 60

High-M(r) mucins [mucin glycoprotein 1 (MG1)] isolated from human saliva from the individual salivary glands were chemically characterized. The carbohydrate content of MG1 derived from palatal (PAL), submandibular (SM) and sublingual (SL) saliva was typical of mucins but showed heterogeneity, especially in the amount of sialic acid and sulphated sugar residues. The physicochemical properties of native MG1s make conventional SDS/PAGE and ion-exchange chromatography unsuitable for investigating differences between individual samples. Recently a density-gradient electrophoresis (DGE) device has been developed, primarily for separation based on the charge of entire cells or cell organelles [Tulp, Verwoerd and Pieters (1993) Electrophoresis 14, 1295-1301]. We have used this apparatus to study the high-M(r) salivary mucins. Using DGE, the MG1s of individual glands were seen to have clearly distinct electrophoretic mobilities, as monitored by ELISA using MG1-specific monoclonal antibodies. Even within a particular MG1 preparation, subpopulations could be distinguished. DGE analysis of a chemically and enzymically modified MG1 series, followed by ELISA and dot-blot detection using specific monoclonal antibodies, lectins and high-iron diamine staining, suggests that the high electrophoretic mobility of PAL-MG1 is mainly the result of a high sulphate content, whereas the SL subpopulations differ mainly in binding type and amount of sialic acid. SM-MG1 most resembles the low-mobility subpopulation of SL-MG1, except that it has a lower sulphate content. In conclusion, DGE appears to be a powerful method for analysis of native mucin; it has been used to demonstrate that MG1s from the various salivary glands are biochemically much more diverse than was previously assumed.
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PMID:Distinct populations of high-M(r) mucins secreted by different human salivary glands discriminated by density-gradient electrophoresis. 763 96


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