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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.
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PMID:A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose. 1246 82

Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.
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PMID:Characterization of the lectin from females of Phlebotomus duboscqi sand flies. 1247 26

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.
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PMID:The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum. 1269 27

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.
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PMID:Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens. 1496 78

The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study. A method was developed for the isolation of the Manila clam lectin (MCL). Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation. SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits. The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography. The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM. MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation. The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc. In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins. MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria. It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans. Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.
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PMID:Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea. 1512 91

Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-(14)C]GalNAc (from UDP-[(14)C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of GIARDIA: This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, D-glucosamine and D-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent K(m) of 0.048 mM for UDP-GalNAc, a V(max) of 0.70 nmol x min(-1) (mg protein)(-1) and a requirement for divalent cations in the following order of preference: Ca(2+), Mg(2+)>Co(2+)>>>Mn(2+), Zn(2+). EDTA inhibits CWS activity.
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PMID:Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall. 1513 86

A preliminary study on humoral defence factors of Indian river prawn, Macrobrachium malcolmsonii was carried out. The serum of animals collected from intermoult stage had an average total protein content of 9.65 g dl(-1) and lysozyme-like activity of 0.04 unit ml(-1). The phenoloxidase activity in haemocyte lysate supernatant was triggered significantly (P < 0.05) by chitosan, Gram-positive and Gram-negative bacterial cells in comparison to other elicitors viz., levamisole, trypsin and glucan. The serum contained an agglutinin against Gram-negative bacteria and a haemagglutinin against a wide range of vertebrate erythrocytes. The haemagglutinin was stable over a wide range of pH (3.0-7.0) and temperatures (-30 degrees C to 60 degrees C). A 413 kDa N-acetyl galactosamine-specific haemagglutinin was purified by single step affinity chromatography and the protein was found to be calcium ion-dependent in nature and made up of five different subunits of varied molecular weights on denaturing SDS-PAGE.
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PMID:Humoral defence factors in Indian river prawn, Macrobrachium malcolmsonii. 1521 34

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with beta-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed beta-D-glucuronic acid and N-acetyl beta-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.
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PMID:Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens. 1576 18

A thorough investigation was conducted for glycoside hydrolase activities in the secreted proteins of Trichinella spiralis. The data demonstrated that the only secreted glycosidase with significant activity was an exo-beta-hexosaminidase with catalysis of the substrates N-acetyl-beta-D-glucosamine, N-acetyl-beta-D-galactosamine and N-acetyl-beta-D-glucosamine-6-sulphate proceeding with an efficiency similar to the human isozyme beta-hexosaminidase A (Hex A). The hydrolysis of N-acetyl-beta-D-glucosamine followed Michaelis-Menten kinetics with a K(m) of 0.187+/-0.025 mM, and catalysis was inhibited competitively by both N-acetyl-beta-d-glucosamine and N-acetyl-beta-D-galactosamine, with K(i) values of 15.75+/-0.99 and 1.17+/-0.24 mM, respectively. The enzyme was maximally active at pH 4.4, had a temperature optimum at 54 degrees C and was thermolabile. We observed no cleavage of N-acetylglucosamine beta1-4 linkages in N-acetylchitooligosaccharides, but significant hydrolysis of N-acetylglucosamine beta1-2 linked to mannose in glycans was detected indicating that the secreted enzyme is linkage specific. The enzyme was partially purified and identified by SDS-PAGE and Western blotting as a protein with an apparent molecular mass of 50 kDa. We established that the protein was glycosylated and showed that the glycan was decorated with tyvelose (3,6-dideoxy-D-arabino-hexose). Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) analysis demonstrated that the carbohydrate moeity was a tyvelose capped tetra-antennary N-glycan corresponding to the structure Tyv(4)Fuc(5)HexNAc(10)Hex(3). All our studies suggest that this is a novel variant of a secreted N-acetyl-beta-hexosaminidase.
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PMID:Characterisation of a secreted N-acetyl-beta-hexosaminidase from Trichinella spiralis. 1624 93

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized D: -lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.
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PMID:Isolation and biochemical characterization of a galactoside binding lectin from Bauhinia variegata candida (BvcL) seeds. 1720 90


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