Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a 'native' molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.
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PMID:Purification and structure of human liver aspartylglucosaminidase. 128 77

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.
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PMID:Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei. 153 12

The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase. The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile. The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues. This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18. The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60. The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units.
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PMID:Isolation and separation of the glycan strands from murein of Escherichia coli by reversed-phase high-performance liquid chromatography. 228 38

The present review summarizes recent data on fat cell adrenoceptors with the aim of clarifying the role played by catecholamines in the regulation of adipocyte metabolism. Part of the review is focused on the possible interest of animal models for the study of catecholamine-mediated effects in human fat cells. It is now clearly demonstrated that human, hamster, dog and rabbit fat cells possess three basic types of adrenoceptor: the beta 1-, alpha 2- and alpha 1-adrenoceptors identified in biological assays or binding studies with selected radioligands. The rat is an exception in the species commonly studied as catecholamines exert an exclusive lipolytic effect through beta-adrenoceptor stimulation, there are no alpha 2-adrenoceptors in rat white fat cells although an alpha 1-adrenoceptor does exist. In human fat cells, physiological amines are lipolytic or antilipolytic. Binding studies have revealed that alpha 2-adrenoceptors are three to four times more numerous than beta 1-adrenoceptors. Moreover physiological amines, in particular epinephrine, have a higher affinity for alpha 2-sites than for beta 1-sites. Dose-response studies of the effect of epinephrine on adenosine-deaminase or isoproterenol-stimulated fat-cells demonstrate an inhibitory effect of epinephrine on lipolysis promoted by stimulation of alpha 2-adrenoceptors which occurs before the commonly described beta 1-adrenergic effect which promotes stimulation of lipolysis. This aspect and its putative physiological interest is described and discussed. Intraspecific variations in adrenergic responses of adipocytes have been briefly analysed. The appearance and disappearance of alpha 2-adrenoceptors according to the extent of adipose tissue and increment of fat cell size are discussed. Variations of adrenergic responsiveness during fasting, calorie restriction or chronic stimulation of the adipocytes by physiological amines are also discussed.
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PMID:Fat cell adrenoceptors: inter- and intraspecific differences and hormone regulation. 299 11

Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3-hydroxymyristic acid. The cell-free preparation also exhibited amidase activity cleaving about 50% of the amide-bound 3-hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester-bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl-beta 1,6-glucosamine disaccharide was not degraded and remained bound to the oligosaccharide part (heptose/3-deoxyoctulosonic acid) of the lipopolysaccharide.
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PMID:In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes. 300 30

Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.
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PMID:Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity. 751 65

We report here the isolation and characterization of a peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the beta-aspartylglycosylamine linkage (GlcNAc beta 1-->Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.
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PMID:A new peptide-N4-(acetyl-beta-glucosaminyl)asparagine amidase from soybean (Glycine max) seeds: purification and substrate specificity. 953 7

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.
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PMID:Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds. 1700 51