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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.
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PMID:N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution. 391 40

Sialyl and galactosyl transferase activities are demonstrated in calf vitreous hyalocytes. For study of sialyl transferase activity, a partially purified vitreous preparation (collagen and hyaluronic acid removed), and bovine submaxillary mucin were treated with an insolubilized neuraminidase before use acceptor of radioactivity from CMP-[3H]-N-acetylneuraminic acid (CMP-[3H]-NAN). For study of galactosyl transferase activity the vitreous preparation was treated first with insolubilized neuraminidase and then with an insolubilized beta-galactosidase before use as acceptor of radioactivity from UDP-[3H]-galactose (UDP-[3H]-gal). Galactosyl transferase requires a divalent metal ion for optimal activity, and the reactions catalyzed by each enzyme are dependent upon pH, time of incubation and concentration of enzyme and/or acceptor.
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PMID:Identification of sialyl and galactosyl transferase activities in calf vitreous hyalocytes. 609 99

Human leucocytes contain two different MU-NANA neuraminidases, which can be distinguished by Concanavalin A binding. The Con A binding form is predominant in lymphocytes (more than 80%) and the non-binding form predominates in granulocytes. The pH optima of both these neuraminidases as well as their subcellular localization as determined by Percoll gradient centrifugation suggest that they are both lysosomal. Immunological studies indicate that the Con A binding form is present in a complex with beta-galactosidase whereas the non-binding form is not. Leucocytes from patients with sialidosis or galactosialidosis are deficient in the Con A binding neuraminidase, whereas the non-binding form is normal. In sialolipidosis both forms are normal. These results demonstrate that leucocytes contain at least two genetically different MU-NANA neuraminidases. Thus, the use of leucocytes should be avoided for the diagnosis of sialidosis and galactosialidosis, and isolated lymphocytes should be used to obtain reliable results.
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PMID:Two genetically different MU-NANA neuraminidases in human leucocytes. 641 40

An adult case of mucolipidosis with beta-galactosidase and neuraminidase deficiencies is reported. The patient was a 35-year-old Japanese female with coarse face, lumbar vertebral beaking, action myoclonus, cerebellar ataxia, clouding of the cornea, macular cherry-red spots, hearing loss and vacuolated lymphocytes, but without mucopolysacchariduria. Her clinical symptoms developed at a late age with a slow progression. The enzyme activities of beta-galactosidase were deficient in leukocytes and cultured skin fibroblasts but normal in serum. Sialic acid-rich glycopeptides and oligosaccharides were increased in the urine. Neuraminidase activities toward fetuin, alpha-N-acetylneuraminosyl-(2 leads to 3) lactose and alpha-N-acetylneuraminosyl-(2 leads to 6) lactose were deficient in cultured fibroblasts. It is suggested that the main disturbance in the present case might be the catabolic process of glycoproteins and oligosaccharides due to neuraminidase deficiency.
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PMID:Adult mucolipidosis with beta-galactosidase and neuraminidase deficiencies. 677 51

Serum resistance of gonococci in most patients is due to sialylation of a Gal beta 1-4GlcNAc group on a conserved 4.5 kDa lipopolysaccharide (LPS) component by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) catalysed by a gonococcal sialyl transferase. This sialylation is enhanced by a low M(r) factor(s) which, like CMP-NANA, is released in diffusates from high M(r) fractions obtained from sonicates dialysed at 4 degrees C. Also, as shown here, this factor(s) is released when the sonicates are dialysed at 18-20 degrees C. The enhancement of sialylation, first demonstrated using enzymes in gonococcal extracts, has been shown to occur in live gonococci and hence probably to have a role in pathogenicity. Gonococci, emerging from lag phase and incubated for 2 h with CMP-14CNANA fixed up to 90% more radiolabel than controls when the second factor(s) was present; their LPS separated by SDS-PAGE contained more radiolabel than control samples and label was not detected in any other component. Fractions with enhancing activity absorbed maximally at about 260 nm but a mixture of UDP-galactose (UDP-Gal), UDP-N-Acetyl galactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc) and UDP-N-Acetyl glucosamine (UDP-GlcNAc) showed no significant enhancing activity. The enhancing action of the low M(r) fractions was unaffected by incubation with beta-galactosidase.
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PMID:Sialylation of lipopolysaccharide by CMP-NANA in viable gonococci is enhanced by low Mr material released from blood cell extracts but not by some UDP sugars. 783 May 28

During short incubations of a Golgi apparatus-enriched subcellular fraction from rat liver with UDP-[3H]GlcNAc, label is efficiently transferred to endogenous acceptors. Most of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that it is mainly associated with N-linked oligosaccharides. The glycoprotein acceptors are resistant to proteases unless detergent is added in amounts greater than the critical micellar concentration. This shows that the acceptors are within the lumen of intact compartments, which have the correct topological orientation expected for the Golgi apparatus in intact cells. Structural characterization of the radiolabeled N-linked oligosaccharides shows a variety of distinct neutral and anionic species. The neutral chains include bi-, tri-, and tetra-antennary molecules with terminal beta-[3H] GlcNAc residues. In vitro sialylation shows that some of the tetra-antennary chains have beta 1,3-linked Gal residues on their unlabeled antennae. An unknown modification appears to block the action of beta-galactosidase on these galactosylated oligosaccharides. Chasing the labeling reaction with a mixtures of UDP-Gal, CMP-Neu5Ac, and adenosine 3'-phosphate,5'-phosphosulfate causes an increase in the percent of radiolabeled anionic oligosaccharides. Most of the negative charge is due to sialic acid (Sia), and some appears to be in phosphodiester-linked [3H]GlcNAc. The sialylated oligosaccharides are a mixture of bi-, tri-, and tetra-antennary species with 1-3-Sia residues, and some of the [3H]GlcNAc residues are directly covered with unlabeled Gal and Sia residues. This in vitro approach should recapitulate reactions that occur in the biosynthesis of N-linked oligosaccharides in the Golgi apparatus of the intact cell. Since the conditions during labeling do not permit inter-compartmental transport, the oligosaccharides produced should represent the biosynthetic capabilities of individual Golgi compartments. Evidence is presented for a functional association of GlcNAc transferases I, II, and alpha-mannosidase II, with separation from GlcNAc transferase IV and/or V. The structures also indicate co-compartmentalization of several GlcNAc transferase(s) with beta-galactosyltransferase(s) and sialyltransferase(s). The compartmental organization of the Golgi apparatus is discussed in light of these findings.
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PMID:Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]N-acetylglucosamine. 834 99

We have investigated the activity of CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the beta-galactosidase alpha-2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activated ras gene decreases the activity of this specific alpha-2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of active O-glycan alpha-2,3-sialyltransferase polypeptides in ras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface of ras-transformed FR3T3 suggesting that no change in the sialylation of O-glycan core 1 appeared in these cells, although the activity of the alpha-2,3-sialyltransferase was decreased.
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PMID:Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: decreased CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase. 835 31

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.
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PMID:Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection. 887 65

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and alpha 2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.
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PMID:Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins. 1046 14

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.
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PMID:The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3. 1060 17


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