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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The levels of the main glycoprotein-derived storage compound, N-acetylglucosamine-asparagine, in various post mortem tissues of three adult patients with inherited deficiency of lysosomal 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria) were measured by gas-liquid chromatography. All aspartylglycosaminuria tissues studied contained significant amounts of N-acetylglucosamine-asparagine, whereas none of the corresponding control tissues contained detectable amounts of this compound. High levels of N-acetylglucosamine-asparagine were found in the liver (3.65 mg/g wet weight), spleen (2.24) and thyroid (2.18), and lower levels in the kidney (0.89), brain (0.53), spinal cord (0.32), sciatic nerve (0.34) and skeletal muscle (0.16). The results show that N-acetylglucosamine-asparagine accumulates chiefly in tissues with important functions in glycoprotein metabolism and/or high endocytic activity. Correlation of the results to the clinical manifestations of aspartylglycosaminuria did not reveal a direct relationship between the amount of N-acetylglucosamine-asparagine stored and the degree of organ dysfunction.
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PMID:N-Acetylglucosamine-asparagine levels in tissues of patients with aspartylglycosaminuria. 744 47

Urinary sialoglycoconjugates were studied in 22 patients with inherited deficiency of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria), in eight obligate heterozygotes, and in age- and sex-matched control subjects. Total sialic acid excretion was significantly higher in the patients (38.3 +/- 17.7 mumol/mmol creatinine, mean +/- S.D.) than in the matched controls (17.7 +/- 7.3 mumol/mmol creatinine, p less than 0.001). The sialic acid output in the heterozygotes did not differ from that of the controls. Gel filtration studies revealed that the increase in urinary sialic acid in aspartylglycosaminuria is of bound type and confined to the low molecular mass region. A linear positive correlation was found between the output of sialic acid and glycoasparagine in the individual patients (r = 0.77, p less than 0.001). The amount of sialylated metabolites excreted in urine did not correlate with the severity of clinical manifestations in aspartyl-glycosaminuria.
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PMID:Urinary sialic acid levels in aspartylglycosaminuria. 747 97

Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
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PMID:Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F. 749 89

Candida albicans and other pathogenic Candida species can use N-acetylglucosamine as a sole carbon source for growth. GlcNAc induces the enzymes of GlcNAc catabolic pathway; besides, under certain conditions, GlcNAc also induces a change from the yeast to germ tube morphology. Glucosamine-6-phosphate deaminase (EC 5.3.1.10) is the terminal enzyme of the GlcNAc catabolic pathway. We have purified the deaminase from C. albicans and studied its characteristics. The size of the deaminase estimated from SDS-polyacrylamide gel electrophoresis is 28 kDa. N-Acetylglucosamine 6-phosphate, an allosteric activator of the Escherichia coli deaminase, has no effect on the activity of the C. albicans enzyme. The deaminase is induced over 100-fold by GlcNAc and its level is about 0.3-0.5% of the proteins in crude extract. Three cDNA clones were obtained from a lambda gt11 expression library by immunoscreening with deaminase antiserum. C. albicans genomic DNA blot hybridization revealed that the NAG1 gene, encoding the glucosamine-6-phosphate deaminase, is present in a single copy. Hybrid-selected translation and immunoprecipitation experiments revealed that the purified deaminase and the protein encoded by the clones were similar in size and in their antigenicity. DNA sequencing revealed that the largest cDNA clone contained the complete open reading frame, which can code for a 27.5-kDa protein. The NH2-terminal sequence (35 residues) determined from the purified deaminase was identical to the sequence of the deduced protein. The Nag1 protein has about 47% identity with the sequence of the E. coli glucosamine-6-phosphate deaminase. Furthermore, RNA blot hybridization showed that GlcNAc induces the expression of NAG1 gene.
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PMID:Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans. 768 45

Aspartylglucosaminidase (AGA: E.C. 3.5.1.26) is a lysosomal amidase that hydrolyzes the N-acetylglucosamine-asparagine linkage as one of the final steps in the breakdown of glycoproteins. Deficiency of this enzyme results in aspartylglucosaminuria (AGU), an inherited lysosomal storage disease. In an attempt to establish the tissue-specific expression of AGA in normal individuals and in AGU patients, we adapted biochemical and immunohistochemical techniques to analyze AGA polypeptides in human cells and tissues. The biochemical analysis revealed the existence of alpha- and beta-subunit structures of AGA in all tissues. Immunohistochemical staining demonstrated a cell specificity in the distribution of AGA: immunoreactivity was strongest in hepatocytes, pyramidal cells in the cerebral cortex, and proximal tubule cells in the kidney. In tissues from AGU patients, AGA immunoreactivity could be detected in hepatocytes and in proximal tubule cells but not in the pyramidal cells. The regulation of the expression of AGA was approached by analyzing the transcript levels and the methylation of the AGA gene. Both heavy methylation of the AGA gene and the constant level of AGA mRNA were typical of a "house-hold" type of enzyme that can be found in small quantities in all tissues. This was in contrast to the variability of the amount of AGA polypeptides observed in different cells and tissues, suggesting that the expression of AGA is regulated not at the transcriptional but rather at the translational level.
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PMID:Expression of aspartylglucosaminidase in human tissues from normal individuals and aspartylglucosaminuria patients. 768 90

N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.
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PMID:Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water. 778 Jan 91

Endo beta-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N'-diacetylchitobiose, N,N'-diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the endo beta-N-acetylglucosaminidase F (Endo F) activity on various free oligosaccharides obtained from the standard glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase) mixture readily cleaved high mannose and complex oligosaccharides (neutral and sialyated) with common core alpha 1-6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core alpha 1-6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endo beta-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides. 806 Jun 71

We previously showed that under defined conditions beta-[3H]funaltrexamine (beta-[3H]FNA) covalently labeled mu-opioid receptors with high specificity in bovine striatal membranes. beta-[3H]FNA-labeled mu-opioid receptors migrated as a broad band with a molecular mass range of 68-97 kDa. It is controversial whether beta-FNA binds irreversibly to mu-opioid receptors in other species. In this study, we demonstrated that beta-[3H]FNA also labeled mu-opioid receptors with high specificity in brain membranes of the guinea pig, rat, and mouse. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed that in each species beta-[3H]FNA specifically bound to a protein in which labeling was greatly reduced by naloxone. These labeled receptors had broad molecular mass ranges, and the molecular masses were different among these species, in the order of cow > guinea pig > rat > mouse. Membranes were subjected to solubilization with 2% Triton X-100 and wheat germ lectin (WGL) affinity chromatography. N-Acetylglucosamine eluted a peak of radioactivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that in all four species the mu receptor was the only protein labeled with beta-[3H]FNA in the WGL eluate. The molecular masses of labeled mu-opioid receptors were 70-88 kDa (median, 77 kDa) for the cow, 66-80 kDa (median, 72 kDa) for the guinea pig, 60-75 kDa (median, 67 kDa) for the rat, and 60-72 kDa (median, 66 kDa) for the mouse. In addition, we investigated the nature of the carbohydrate moieties linked to the receptor protein and whether the species variation in the molecular mass was due to variable degrees of glycosylation. The bovine WGL eluate was treated with various glycosidases. Neuraminidase treatment decreased the receptor molecular mass by 6-7 kDa, whereas alpha-mannosidase had no effect. Removal of N-linked carbohydrates at asparagine residues by peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase (N-Glycanase) resulted in a much sharper specifically labelled protein band of 43 kDa. These results indicate that mu-opioid receptors are heavily glycosylated and the major carbohydrate moieties are of the complex type, N-linked to asparagine. After the WGL eluates for the four species were treated with N-Glycanase, the labeled receptors became much sharper bands with very similar molecular masses, i.e., 43 kDa for the cow and guinea pig, 39 kDa for the rat, and and 40 kDa for the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Beta-[3H]funaltrexamine-labeled mu-opioid receptors: species variations in molecular mass and glycosylation by complex-type, N-linked oligosaccharides. 823 25

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.
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PMID:The biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked and O-linked glycans labeled by UDP-[6-3H]N-acetylgalactosamine. 834 1

Glycosylasparaginase (EC 3.5.1.26) is an amidase, which cleaves the N-glycosidic linkage during glycoprotein degradation leading to the liberation of L-aspartic acid from various glycoasparagines. In this work we demonstrate that glycosylasparaginase is also capable of catalyzing the synthesis of the N-glycosidic bond by N-beta-aspartylation of beta-glycosylamine using 1-amino-N-acetylglucosamine as the nucleophile and L-aspartic acid beta-methyl ester as the beta-aspartyl donor. Kinetic studies indicated that beta-glycosylamine has 1390-fold higher reactivity than water in the de-beta-aspartylation of the beta-aspartylenzyme, indicative of the presence of a beta-glycosylamine binding sub-site at the substrate binding site of glycosylasparaginase. The reaction can be applied to glycosylaparaginase-catalyzed biosynthesis of novel glycoasparagines.
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PMID:Enzymatic synthesis of the N-glycosidic bond by beta-aspartylation of glycosylamines. 856 87


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