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 studied the differential susceptibility to N-glycanase (peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase) of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue or hypothyroid pituitary tissue were incubated with D-[2-3H]mannose for 6 h. [3H]Mannose-labeled TSH or free alpha-subunits were obtained from homogenates using specific antisera and were digested with N-glycanase in their native state or after heat denaturation and reduction in the absence or presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that the effects of N-glycanase at the individual glycosylation sites could be determined. N-Glycanase treatment of native molecules did not cleave oligosaccharides efficiently at Asn56 of alpha-subunits and Asn23 of TSH beta, whereas oligosaccharides at Asn82 of alpha-subunits were more susceptible regardless of whether the alpha-subunits were combined with TSH beta. Heat denaturation, reduction, and the presence of detergents did not substantially increase the cleavage by N-glycanase of the protected oligosaccharides, suggesting that the primary structures of the TSH subunits influenced efficiency at specific sites. Pretreatment of free alpha-subunits with trypsin failed to enable N-glycanase to work fully, as oligosaccharides at Asn56 were cleaved less effectively than those at Asn82. Thus, the susceptibility to N-glycanase differs at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these differences may result from effects of the primary structures of the TSH subunits.
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PMID:Differential susceptibility to N-glycanase at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits. 245 9

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Partial purification and characterization of sialate O-acetylesterase from bovine brain. 277 45

Down-regulation ("curb") of hexose transport in Chinese hamster lung fibroblasts has been studied in a metabolic mutant highly defective in phosphoglucose isomerase (PGI; glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). In the parental strain (PGI+) glucose as well as glucosamine and mannose were able to elicit a curb of the hexose transport system. In the PGI mutant, only glucose was able to mediate a transport curb. The inability of glucosamine and mannose to promote a transport curb in the PGI strain must be ascribed to the fact that the 6-esters of these aldohexoses are converted by their own specific deaminase and isomerase to fructose 6-phosphate, which initiates the pyruvate-tricarboxylate energy-yielding pathway but cannot be converted to glucose 6-phosphate in the mutant. The latter ester can be metabolized, but its metabolism in the mutant is confined to the pentose shunt. It is shown that inhibitors such as 2,4-dinitrophenol and malonate exert only slight inhibition of the pentose shunt yet release the glucose-mediated curb elicited by glucose and glucosamine in the parental PGI+ strain and also the glucose transport curb persisting in the PGI mutant.
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PMID:Down-regulation of the hexose transport system: metabolic basis studied with a fibroblast mutant lacking phosphoglucose isomerase. 695 19

Aspartylglucosaminuria (McKusick 208400) is a lysosomopathy associated with aspartylglucosaminidase (L-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) deficiency. It has been most frequently encountered in Finland, where the regional incidence may be as high as 1 in 3600 births. In North America it is very rare, having been reported in only 8 patients. We encountered 4 patients with aspartylglucosaminuria in a Canadian family of 12 siblings. The 4 siblings affected--2 brothers and 2 sisters--were apparently normal at birth; however, their developmental milestones, particularly speech, were slow, and they acquired only a simple vocabulary. Throughout life, there was a progressive coarsening of facial features; 3 had inguinal hernia and recurrent diarrhea; all became severely retarded and by the 4th decade showed evident deterioration of both cognitive and motor skills; 2 exhibited cyclical behavioural changes. Three of the siblings have died, at 33, 39 and 44 years of age. Two died of bronchopneumonia and 1 of asphyxiation following aspiration. In the urine of all 4 siblings, and in the 1 liver examined, we found 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glucosamine (GlcNAc-Asn) and alpha-D-mannose-(1,6)-beta-D-mannose-(1,4)-2-acetamido- 2-deoxy-beta-D-glucose-(1,4)-2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta - D-glucosamine (Man2-GlcNAc2-Asn). Compared with the level of activity in controls, aspartylglucosaminidase activity was less than 2% in fibroblasts from 3 of the siblings, less than 0.5% in leukocytes from 1 sibling, and less than 1% in the liver of 1 sibling, whereas other acid hydrolase activities in these tissues were normal. Ultrastructural studies of skin showed that fibroblasts, endothelial cells and pericytes contained vacuoles with fine reticulo-floccular material. Glial and neuronal cells of the central nervous system showed similar inclusions as well as others composed of concentric or parallel membranous arrays intermingled with lipid droplets.
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PMID:Aspartylglucosaminuria in a Canadian family. 962 65

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.
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PMID:A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups. 1560 34

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.
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PMID:Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase. 1730 46

Cultural practices for canopy management in grapevines rely on intensive manipulation of shoot architecture to maintain canopy light levels. In contrast to common model plant systems used to study regulation of branch outgrowth, the grapevine has a more complex architecture. The node contains first, second and third order axillary meristems. The prompt bud (N+1) develops into a summer lateral and a latent compound bud develops in the basal node of the summer lateral (N+2, N+3(1,2)). The outgrowth potential of latent buds was determined using common canopy management treatments (shoot tip decapitation and removal of summer laterals and leaves) and monitoring the rate of latent bud outgrowth. Two shoot node regions (apical and basal) with differential outgrowth potential were characterized and it was noted that the shoot tip, summer laterals and leaves in addition to node position contributed to the inhibition of latent bud outgrowth. To advance the understanding of the molecular regulation of bud outgrowth and paradormancy in the complex shoot architecture of grapevines, the expression of auxin and cytokinin genes involved in branching (amidase (VrAMI1), PINFORMED-3 (VrPIN3) and isopentenyl transferase (VrIPT)) were monitored in shoot tips and differentially aged buds of Vitis riparia grapevine shoots. In addition, Histone 3 (VrH3) and a hexose transporter (VrHT1) expression were monitored as a measure of tissue activity. The expression of VrAMI1 and VrPIN3 remained constant in actively growing shoot tips and decreased significantly with increasing bud maturation in paradormant buds. VrHT1 expression was greater in buds than in any other plant tissue tested. VrHT1 may have the potential to be used as an indicator of paradormancy status in grapevines. These characterizations in the complex architecture of the grapevine provide an excellent model system for molecular analysis of bud outgrowth and shoot architecture development.
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PMID:Auxin and cytokinin related gene expression during active shoot growth and latent bud paradormancy in Vitis riparia grapevine. 2232 93