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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.
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PMID:Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins. 172 51

The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3289-3293]. This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide: N-glycosidase F (PNGase F) or endo-beta-N-acetylglucosaminidase H digestion of glycoproteins. Separations were carried out at a constant base concentration (0.1 M NaOH) using linear gradients from 0 to 0.2 M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating "oligosaccharide maps" of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering salt-free oligosaccharides after this chromatography were also investigated. On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of 0.15-0.2 M at a flow-rate of 1 ml/min. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversion to permethyl derivatives or after reductive amination with rho-aminobenzoic acid ethyl ester.
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PMID:Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography. 232 8

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

Capillary electrophoresis with laser-induced fluorescent detection, a one-dimensional version of the well-established planar analytical method of polyacrylamide gel electrophoresis, has been proven to be a powerful new microanalytical method for profiling complex carbohydrates. In this paper a comparison is presented between the planar high concentration polyacrylamide gel electrophoresis method and capillary electrophoresis of different carbohydrates with respect to performance and efficiency. N-Linked oligosaccharides were released from several glycoproteins, including fetuin, human immunodeficiency virus (HIV) envelope recombinant glycoprotein (GP-120), alpha 1-acid glycoprotein and ribonuclease B, using recombinant peptide-N-glycosidase F (PNGase F). Both separation methods involve labeling of the released carbohydrates at the reducing end with the fluorescent dye, disodium 8-amino-1,3,6-naphthalene trisulfonate (ANTS). Fluorophore labeling was followed by separation of the labeled oligosaccharides either by high concentration polyacrylamide gel electrophoresis or capillary electrophoresis.
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PMID:Capillary and slab gel electrophoresis profiling of oligosaccharides. 749 47

We evaluated two independent models of eosinophil differentiation for their ability to synthesize the ribonuclease toxins eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Cells from the clone 15 subline of HL-60 (human promyelocytic leukemia) produced both EDN and ECP; production of EDN increased in response to butyric acid (BA). CD34+ peripheral blood progenitor cells (PBPCs) grown with cytokines promoting eosinophil differentiation also produced EDN. EDN from both the clone 15 and PBPCs was more heterogeneous and heavily glycosylated (approximately 22-45 kDa) than EDN from the mature peripheral blood eosinophils (18-25 kDa). The heterogeneity of EDN from the clone 15 cells was not altered by endoglycosidase Hf, whereas treatment with peptide-N-glycosidase F (PNGase F) produced a single-band immunoreactive band (approximately 15 kDa). In contrast, only the highest molecular weight forms of EDN from differentiated PBPCs were eliminated by PNTGase F (reduced to 22-35 kDa), suggesting the presence of uncharacteristic forms of posttranslational modification. Synthesis of hyperglycosylated proteins has not been previously reported in PBPCs and is a feature shared with tumor cells and cell lines.
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PMID:Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor cells. 761 5

The structures of ribonuclease B oligosaccharides have previously been shown to be high mannose type by methylation analyses and sequential exoglycosidase digestion. Due to the unique nature of these oligosaccharides, in that all mannosyl residues are attached by alpha-(1-->2)-linkages beyond the branch points, methylation analysis fails to solve the exact structures beyond Man5. Therefore, we have undertaken this study using 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In this study, bovine pancreatic ribonuclease B was first reduced and carboxymethylated, and was then deglycosylated by peptide/N-glycosidase F (PNGase F). The released oligosaccharides were fractionated by Bio-Gel P-4 chromatography to give five pools, Man5 through Man9. The structures of the oligosaccharide pools were then studied by laser desorption time of flight mass spectrometry and 1H NMR spectroscopy at 300 MHz. For Man5, Man-A and Man-B are attached in alpha-(1-->3)- and alpha-(1-->6)-linkages to the alpha-(1-->6)-linked Man-4' of the pentasaccharide core structure. For Man6, Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man7 exists as three structural isomers, and has the additional mannosyl residue (Man-D) linked alpha-(1-->2) to Man-A, Man-B, and Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man-7 exists as three structural isomers, with the additional two mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C. For each position, Man-A, Man-B, and Man-C, the extent of occupancy by one of the additional alpha-(-->)-linked mannosyl residues was 15, 94, and 91%, respectively. Man9 is a single component, with the three additional mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C, respectively. The relative molar proportions of Man5 to Man9 are 57, 31, 4, 7, and 1%, respectively. This report presents for the first time the complete structural characterization of the oligosaccharides from ribonuclease B.
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PMID:A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry. 795 10

The N-glycans have been removed by peptide-N-glycosidase F (PNGase F) from purified human non-secretory RNases derived from kidney, liver and spleen. The spleen RNase was purified by two procedures, one of which did not include the usual acid treatment step (0.25 M H2SO4, 45 min, 4 degrees C), to determine if acid treatment alters the carbohydrate moieties. The N-glycans of the RNases were fractionated by Bio-Gel P-4 chromatography and analysed by 600 MHz 1H-NMR spectroscopy and electrospray mass spectrometry. All four non-secretory RNase preparations contained the following structures: [formula: see text] The relative amounts of the trisaccharide, pentasaccharide and hexasaccharide appeared to vary slightly in the different tissue RNases. The overall results indicate: (i) that acid treatment during purification does not alter the N-glycans of non-secretory RNases; (ii) that the N-glycans from kidney, liver and spleen non-secretory RNases are very similar, if not identical, to one another, but different from the N-glycan structures reported for secretory RNase.
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PMID:Human non-secretory ribonucleases. II. Structural characterization of the N-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry. 835 50

Calnexin is a membrane protein of the endoplasmic reticulum that associates transiently with newly synthesized N-linked glycoproteins in vivo. Using defined components, the binding of ribonuclease B (RNase B) Man7-Man9 glycoforms to the luminal domain of calnexin was observed in vitro only if RNase B was monoglucosylated. Binding was independent of the conformation of the glycoprotein. Calnexin protected monoglucosylated RNase B from the action of glucosidase II and PNGase F but not from that of Endo H, which completely released the protein from calnexin. These observations directly demonstrate that calnexin can act exclusively as a lectin.
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PMID:Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. 901 2

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated with the heavy chains.
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PMID:Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. 923 2

A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.
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PMID:Purification and characterization of glycolactin, a novel glycoprotein from bovine milk. 1073 13


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