Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed. It shows limited activity with two intact glycoproteins, ribonuclease B and yeast invertase, and gives optimal rate with glycopeptides. Free glycan-Asn derivatives are poor substrates in comparison with glycopeptides or glycan-Asn derivatives where the alpha-amino group has been dansylated. The amidase will liberate both high mannose, hybrid, and asialo-complex glycans from both proteins and peptides, but many glycans in intact proteins or in long peptides are resistant to the amidase and become active as substrates only after further proteolytic cleavage. The best substrates appear to be those with the glycosylated asparagine no more than 4-5 residues in from either the NH2- or COOH-terminal end of the peptide. Sialylated glycans do not appear to be released by the amidase.
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PMID:Purification and characterization of two glycopeptide hydrolases from jack beans. 333 94

A novel endo-beta-N-acetylglucosaminidase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-beta-N-acetylglucosaminidase, Endo-M, could liberate the complex type of asparagine-linked oligosaccharides by hydrolysis of diacetylchitobiose linkage from glycoproteins. The treatment of Endo-M with N-acetyl-glucosamine and asialotransferrin glycopeptide having the complex type of oligosaccharides resulted in the transfer of the released oligosaccharide from the glycopeptide to N-acetyl-glucosamine. The structure of the product after transfer was deduced to be (GlcNAc)2-Man-(Gal-GlcNAc-Man)2 by a combination method of pyridylamination and high performance liquid chromatography, and mass-spectrometry. The enzyme could transfer the complex type of oligosaccharide from asialotransferrin glycopeptide to bovine ribonuclease with the high-mannose type of oligosaccharide. This will lead to the construction of neoglycoproteins containing different types of oligosaccharides.
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PMID:Transglycosylation activity of Mucor hiemalis endo-beta-N-acetyl-glucosaminidase which transfers complex oligosaccharides to the N-acetylglucosamine moieties of peptides. 807 62

High-performance liquid chromatography of carbohydrate materials on graphitized carbon columns (GCC) has some advantages over other types of chromatography. Oligosaccharides and glycopeptides with few amino acids are barely retained on reversed-phase columns even under high salt or low pH conditions, but can be retained effectively on a graphitized carbon column. Moreover, elution of GCC requires concentrations of organic solvents lower than that required for normal-phase columns. The usefulness of graphitized carbon columns is exemplified by the following results: (i) Man9GlcNAc2 with only Asn or Asn-Phe (derived from soybean agglutinin) was not retained by a C18 reversed-phase column, but could be separated on a GCC with a gradient of 10-45% CH3CN in 30 min. (ii) Ribonuclease B glycopeptides obtained by Pronase digestion could be separated on GCC with a gradient of 10-30% CH3CN, but they were not retained on a C18 reversed-phase column even with water as eluent. (iii) Oligosaccharides released from ribonuclease B by endo-beta-N-acetylglucosaminidase were separated from each other and peptides on GCC with a linear gradient of 10 mM NH4OH to 10 mM NH4OH-12.5% CH3CN in 50 min at 70 degrees C. Silica-based columns do not allow such an alkaline eluent. (iv) Chito-oligosaccharides (DP 1-9) are well separated within 40 min on GCC with a gradient (10 mM NH4OH-10 mM NH4OH with 25% CH3CN) at 50 degrees C. Chito-oligosaccharides could not be separated by high-performance anion exchange columns such as Carbopac PA-1.
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PMID:High-performance liquid chromatography of glycopeptides and oligosaccharides on graphitized carbon columns. 808 79

The enzyme that catalyzed the conversion of human salivary alpha-amylase family A (HSA-A) to family B (HSA-B) was identified. It was partially purified from the precipitate obtained by centrifugation of human saliva at 105,000 x g for 60 min by solubilization with 3[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate and column chromatographies with Sephacryl S-300-HR and hydroxylapatite. The enzyme preparation was practically free from contaminating exoglycosidases and proteases. The enzyme cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of HSA-A, as shown by the isolation of the protein moiety which contained 1 GlcNAc and 1 Fuc residue and the sugar chain (Gal)2(Fuc)1(GlcNAc)2(Man)3(GlcNAc). This enzyme also cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of human transferrin tetraglycopeptide Asn-Tyr-Asn(GlcNAc)2(Man)3(GlcNAc)2(Gal)2-Lys to yield equimolar amounts of peptide Asn-Tyr-Asn(GlcNAc)Lys and sugar chain (Gal)2(GlcNAc)2(Man)3(GlcNAc). The enzyme was identified as an endo-beta-N-acetylglucosaminidase. The enzyme acted on HSA-A with desialylated and defucosylated outer chain moieties of the sugar chains at a similar rate as that of native HSA-A. The enzyme activity was reduced to 13 and 5% using HSA-A with the sugar chains whose outer chain moieties lacked Gal and GlcNAc, respectively, from the nonreducing end. The enzyme also acted on human transferrin, calf fetuin, and asparagine oligosaccharides of transferrin and fetuin. On the other hand, the enzyme did not act on ovalbumin, RNase B, Taka-amylase, yeast invertase, and ovalbumin asparagine oligosaccharides. These results indicate that human salivary endo-beta-N-acetylglucosaminidase is specific for complex type sugar chains and can release the sugar chains from native glycoproteins and glycopeptides regardless of the existence of a Fuc residue on the proximal GlcNAc of the N,N'-diacetylchitobiose core of their sugar chains. The source of the enzyme was epithelial cells peeling from the oral cavity epithelium into saliva. The enzyme was thought to be integrated on the surface of the epithelial cell membrane. This enzyme was named endo-beta-N-acetylglucosaminidase HS. Thus, these studies indicate that the properties of the enzyme are distinct from those of known endo-beta-N-acetylglucosaminidase and endo-beta-N-acetylglucosaminidase HS is a novel endo-beta-N-acetylglucosaminidase.
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PMID:Human salivary endo-beta-N-acetylglucosaminidase HS specific for complex type sugar chains of glycoproteins. 834 Apr 28

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.
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PMID:Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A. 1062 87

Arthrobacter protophormiae produced a high level of extracellular endo-beta-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-beta-N-acetylglucosaminidase was very similar to that of Endo-C(II) from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.
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PMID:Induction and Purification of Endo-beta-N-Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin. 1634 72

An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core by use of azido-containing Man3GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of alphaGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications.
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PMID:Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands. 1880 85