Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucosamine-binding sites were detected in Lowicryl K4M-embedded guinea pig middle ear mucosa by electron microscopy, using glucosaminyl bovine serum albumin. Incubation of ultrathin tissue sections with gold-labeled glucosaminyl bovine serum albumin (GlcN/BSA/gold) resulted in binding mainly on cilia, microvilli, rough endoplasmic reticulum and nuclei. The sugar binding was not inhibited after ultrathin sections had been digested with trypsin or neuraminidase. Various carbohydrates and glycoconjugates were tested as competitive inhibitors of GlcN/BSA/gold labeling on the tissue sections. The sugar specificity range detected by the glucosamine-binding sites included glucosamine, N-acetylglucosamine, mannose and fucose, whereas N-acetylgalactosamine, galactose and glucose were not detectable. A series of endotoxic substances such as Salmonella minnesota Re595 lipid A complex with BSA and lipopolysaccharides (LPS) derived from Escherichia coli 055:B5 or S. minnesota Re595 also competed with GlcN/BSA/gold binding. This indicates that the lipid A backbone glucosamine or other carbohydrate portions of LPS is a part of the structure recognized by glucosamine-binding sites.
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PMID:Sugar-binding sites with specificity for glucosamine in the guinea pig middle ear mucosa. 828 7

The highly glycosylated domains of rat small intestinal mucins were isolated after reduction and trypsin digestion and separated into two populations (A and B) by gel chromatography. The molecular mass values were 650 and 335 kDa, respectively, and the relative yields suggest that the two glycopeptides occur in equimolar proportions. Electron microscopy revealed linear structures with weight average lengths of 230 nm (A) and 110 nm (B) corresponding to a mass/unit length of about 3 kDa/nm. The protein cores (17-19%) contain large amounts of threonine (over 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfate account for approximately 80 and 0.5%, respectively, and gas chromatography-mass spectrometry showed that the patterns of neutral and sialic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc residues, the average oligosaccharide is about 4 sugar residues long, and the largest species observed are heptasaccharides. The major neutral and sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)GalNAcol, respectively. Sialic acid is present as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extractions of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas exposure to dithiothreitol or high speed homogenization accomplished complete solubilization. The "subunits" obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments corresponding in size to the latter are obtained after cleavage with trypsin. Most of the mucins from rat small intestine thus occurs as an insoluble glycoprotein complex composed of subunits joined with disulfide bonds. The subunits contain two highly glycosylated regions with different lengths substituted with very similar oligosaccharides.
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PMID:Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine. 836 Jan 70

The equine embryonic capsule replaces the zona pellucida and envelopes the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35-40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric assays and high-performance anion-exchange chromatography also showed it to have mucin-like characteristics. Gal, GalNAc, sulfated sugars, and sialic acid make up a high proportion of the capsular carbohydrate, while GlcNAc, Glc, and Man are minor components. These findings were verified using lectin histochemical staining of frozen sections of conceptuses. The results of amino acid analysis were also consistent with the proposal that the capsular glycoproteins belong to the mucin family. Removal of the covalently bound carbohydrate by beta-elimination under reducing conditions demonstrated that the capsule is O-glycosylated mainly on threonine residues. Affinity chromatography on jacalin-agarose confirmed that, like mucins, the capsular glycoproteins are heavily O-glycosylated. SDS-PAGE analysis revealed a prominent 21-kDa band, specific to the capsule, in preparations solubilized by trypsin but not by other proteases. Characterization of its constituent glycoprotein(s) should be helpful in elucidating the role of the capsule (and analogous blastocyst coverings in other species) during early pregnancy.
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PMID:Mucin-like glycoproteins in the equine embryonic capsule. 847 Dec 47

A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-[32P]ATP (Pastuszak, I., Drake, R., and Elbein, A. D. (1996) J. Biol. Chem. 271, in press). A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15. To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared. The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations. Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc. On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc. These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations. This transfection thus allows the yeast mutant to grow slowly on galactose-containing media.
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PMID:Identification of the GalNAc kinase amino acid sequence. 879 85

Three monoclonal antibodies (mAbs), M344, M300 and M75, were shown to define a unique tumour-associated antigen (TAA) of superficial bladder tumours. The antigenic determinants are expressed on a very-high-molecular-mass component and, in about 50% of the positive samples, one determinant is also detected on a 62 kDa molecular species, observed only under reducing conditions. The objectives of the present study were to characterize further this TAA by analysing (1) the biochemical nature of the epitopes recognized by the three mAbs, and (2) the biochemical and structural features of the molecule bearing them. The antigenicity was resistant to heat denaturation, trypsin and alpha-chymotrypsin treatments but highly sensitive to papain and Pronase digestion. NaIO4 oxidation decreased reactivity to mAbs M344 and M300 but enhanced reactivity to mAb M75. The three determinants were insensitive to beta-galactosidase and alpha-L-fucosidase but were sensitive to Vibrio cholerae neuraminidase. None of the three mAbs reacted with ovine, bovine or porcine submaxillary mucins. Deglycosylation with O-glycosidase or trifluoromethanesulphonic acid completely abolished the reactivity of the mAbs whereas N-glycosidase F deglycosylation had no appreciable effect. The presence on the molecule of cryptic Gal(beta(1-3))GalNAc as a major core disaccharide was demonstrated by a heterologous sandwich assay using mAb M75 and peanut agglutinin. Thiol reduction using beta-mercaptoethanol increased mobility of the high-molecular-mass component in polyacrylamide gels. We thus conclude that mAbs M344 and M300 react with sialylated carbohydrate epitopes, and mAb M75 reacts with a partially cryptic and periodate-resistant sialylated epitope expressed on a typical secreted high-molecular-mass oligomeric mucin which we named MAUB for mucin antigen of the urinary bladder.
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PMID:Biochemical analysis of a bladder-cancer-associated mucin: structural features and epitope characterization. 903 80

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.
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PMID:Proteolytic release and partial characterization of human sperm-surface glycopeptides. 924 37

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc.
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PMID:Cell-surface carbohydrates of Entamoeba invadens. 934 47

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.
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PMID:Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity. 953 10

This study was performed to analyze the structural variety of O-glycans on the IgA1 hinge in IgA nephropathy (IgAN). The IgA1 fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated from 13 IgAN patients, eight healthy control subjects, and 11 patients with other primary glomerulonephritides by pyridylethylation, trypsin treatment, and Jacalin affinity chromatography. Because of the use of Jacalin, only the Gal beta 1-3GalNAc residue containing IgA was analyzed. The molecular weights (MW) of the IgA1 fragments treated by the following sequential treatment by exoglycosidases were estimated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: (1) Sialidase treatment: the MW of the two observed peaks A and B were compatible with (A) HP + 4GalNAc + 4Gal and (B) HP + 5GalNAc + 4Gal. (2) Sialidase and galactosidase: the MW of the two identified peaks a and b were consistent with (a) HP + 4GalNAc and (b) HP + 5GalNAc. (3) Sialidase, galactosidase, and alpha-N-acetylgalactosaminidase. All subjects revealed one peak, indicating the 33-mer IgA1 hinge peptide core. The intensity rate of peak B/A was significantly decreased in the IgAN group (mean +/- SD, 1.01 +/- 0.08) compared with the negative control subjects (healthy group, 1.15 +/- 0.06, P = 0.0048; other glomerulonephritis group, 1.13 +/- 0.10, P = 0.0049; Scheffe's F test). These results suggested the presence of a defect in the Gal and/or GalNAc residues in the IgA1 hinge glycopeptides in IgAN.
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PMID:Analyses of IgA1 hinge glycopeptides in IgA nephropathy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 955 59

To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.
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PMID:Binding characteristics of an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132). 1042 83


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