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Biomedical subjects

R Geyer

Publications and source records attributed to R Geyer.

At least 127 records · Page 7Linked to original sources
PubMed1984

Major oligosaccharides in the glycoprotein of Friend murine leukemia virus: structure elucidation by one- and two-dimensional proton nuclear magnetic resonance and methylation analysis.

The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues.

Acetylglucosaminidase↗
PubMed1984

Structure of the oligosaccharides sensitive to endo-beta-n-acetylglucosaminidase H in the glycoprotein of Friend murine leukemia virus.

The surface glycoprotein (mixture of isoglycoproteins with Mr 69 000 and 71 000) was isolated from the particles of Friend murine leukemia virus, and was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. Roughly 20% (w/w) of the carbohydrates in this glycoprotein were thus released, and they were fractionated by high-performance liquid chromatography after reduction with KB3H4/NaBH4. The radioactive oligosaccharitol fractions obtained were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by fast atom-bombardment mass spectrometry, as well as by capillary gas-liquid chromatography/mass spectrometry following hydrolysis, reduction and peracetylation. Around 85% (mol/mol) of the endo-H-sensitive viral glycans were thus found to be oligomannosidic oligosaccharitols of size classes Man5GlcNAcOH, Man6GlcNAcOH, Man8GlcNAcOH, Man7GlcNAcOH, and Man9GlcNAcOH (in order of prevalence), and the major structural isomers of each size class were identified. About another 15% (mol/mol) of the oligosaccharitols were shown to be of the 'mixed type', comprising mainly four species in which the Man(alpha 1----6)-branch of the Man alpha 1----6 (Man alpha 1----3) Man beta 1----4GlcNAcOH core is substituted by one or two additional alpha-mannoses, while the Man(alpha 1----3)-branch carries an N-acetyllactosamine unit substituted by sialic acid, or by Gal(alpha 1----3).

Acetylglucosaminidase↗
PubMed1984

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12.

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.

Amino Acid Sequence↗
PubMed1984

Structure of the major oligosaccharides in the fusion glycoprotein of Newcastle disease virus.

The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.

Acetone↗
PubMed1984

Structural studies on the major oligosaccharides in a variant surface glycoprotein of Trypanosoma congolense.

The carbohydrate moieties in the four isotypes of a variant surface glycoprotein from Trypanosoma congolense were analyzed. All variant surface glycoprotein isotypes were found to contain up to 15% by weight of D-galactose, D-mannose, and N-acetyl-D-glucosamine in molar ratios approaching 1:3.2:3.9 (isotypes I-III) or 1:2.4:2.4 (isotype IV); in addition, the presence of sialic acid could be demonstrated. After metabolic labelling with D-[6-3H]glucosamine, the four isoglycoproteins were successively digested with pronase and with endo-beta-N-acetylglucosaminidase H. Up to two thirds of the oligosaccharides were thus liberated and were separated by gel filtration, and by high performance liquid chromatography. Using methylation, gas chromatography, mass spectrometry and digestion with alpha-mannosidase, they were shown to be mainly typical oligomannosidic oligosaccharides of size classes Man5GlcNAc to Man9GlcNAc. The residual glycans were liberated by hydrazinolysis, and were fractionated by serotonin affinity chromatography. After separation by gel filtration, the neutral oligosaccharides from isotype I were subjected to methylation analysis and successive exoglycosidase digestions. They were found to be biantennary oligosaccharides of the N-acetyllactosaminic type: (GalGlcNAc)2Man3GlcNAc1-2. Only about 30% of the sialylated glycans were susceptible to neuraminidases. The T. congolense variant surface glycoprotein studied here contains mainly high mannose and biantennary 'complex' oligosaccharides as found in many other eukaryotic glycoproteins, except that they seem to carry unusually substituted/linked sialic acid residues.

Animals↗
PubMed1984

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1.

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

Carbohydrate Sequence↗
PubMed1983

Separation and sugar component analysis of the oligosaccharides in the surface glycoproteins of Newcastle disease virus.

The precursor glycoproteins HN0 and F0 in the surface spikes of Newcastle Disease Virus strain Ulster as produced by MDBK cells, were found to contain 10.4 and 11.9 weight per cent, respectively, of the sugars typical for N-glycosidically linked glycoprotein glycans. A molar ratio of D-mannose:D-galactose: L-fucose:N-acetyl-D-glucosamine approaching 1.0:1.1:0.5:1.0 was found for HN0, and of 1.0:0.7:0.3:0.6 for F0. By a sequence of degradation (with pronase, with endo-beta-N-acetylglucosaminidase H [endo H], and by hydrazinolysis) and separation procedures (Concanavalin A-affinity and Biogel P-4 chromatography), the radiolabelled carbohydrate moieties of NDV HN0 and F0 (as oligosaccharitols) were separated into (at least) ten and eight fractions, respectively. Separate in vivo labelling with tritiated derivatives of the four sugars showed that both glycoproteins contain oligosaccharides of the oligomannosidic ("high mannose"), of the N-acetyllactosaminic ("complex"), as well as of the "mixed" type. The majority of the oligosaccharides in F0, but not of those in HN0, was found to be endo H-sensitive.

Carbohydrates↗
PubMed1983

Methylation analysis of complex carbohydrates in small amounts: capillary gas chromatography-mass fragmentography of methylalditol acetates obtained from N-glycosidically linked glycoprotein oligosaccharides.

A version of the methylation analysis of complex carbohydrates by gas chromatography-mass spectrometry of the methylalditol acetates (H. Björndal, C. G. Hellerquist, B. Lindberg, and S. Svensson (1970) Angew. Chem. 82, 643-674) is described. With this version 100- to 500-pmol samples of N-glycosidically linked glycoprotein oligosaccharides may be analyzed. The method is based on the use of capillary columns which allow the separation of all partially methylated alditol acetates potentially obtained from this group of oligosaccharides and on their selective and sensitive detection by mass fragmentography after chemical ionization with ammonia.

Chromatography, Gas↗
PubMed1983

Detection and analysis of aminoparathion in human postmortem specimens.

Postmortem samples from two fatalities involving parathion ingestion were examined. Parathion could not be detected in the liver tissue but a significant quantity of a related compound was detected. This was shown to be aminoparathion, a biotransformation product of parathion. The substances were extracted with hexane and analyzed by gas-liquid chromatography on a 3% OV-210 column. Thin-layer chromatography and mass spectrometry were also performed. The transformation of parathion to aminoparathion in human tissue has not been previously reported.

Biotransformation↗
PubMed1982

Separation procedure and sugar composition of oligosaccharides in the surface glycoprotein of Friend murine leukemia virus.

The sugar composition of the surface glycoprotein from Friend murine leukemia virus was determined by gas-liquid chromatography of the alditol acetates and by the thiobarbituric acid method, respectively. N-Acetylglucosamine, mannose, galactose, sialic acid and fucose were found in a molar ratio around 15.2:11.6:7.4:3.3:1.0. Ten oligosaccharide fractions were obtained from glycoprotein preparations by a suitable sequence of degradation (with pronase, endo-beta-N-acetylglucosaminidase H, neuraminidase, and by hydrazinolysis) and separation procedures (concanavalin A-affinity chromatography and gel filtration). The qualitative sugar composition of these fractions was analyzed by in vivo labelling with D-[6-(3)H]glucosamine, D-[2-(3)H]mannose, D-[6-(3)H]galactose, or L-[6-(3)H]fucose, and their molecular weights were estimated from the gel elution volumina. Four fractions of N-glycosidically linked oligosaccharides of the oligomannosidic ('high mannose') type oligomannosidic7-oligomannosidic10, about seven to ten sugar residues), two of the mixed (M11 and M12), and four of the N-acetyllactosaminic ('complex') type (N-acetyllactosaminic9, probably nine sugar residues; N-acetyllactosaminica-N-acetyllactosaminic c, size unknown) were thus identified.

Chromatography, Affinity↗
PubMed1979

A lipopolysaccharide-binding cell-surface protein from Salmonella minnesota. Isolation, partial characterization and occurrence in different Enterobacteriaceae.

1. Protein extracts obtained from Salmonella minnesota Re mutant cells by treatment with EDTA/NaC1 solution contain a protein which exhibits high affinity to bacterial lipopolysaccharides. The isolation and partial characterization of this lipopolysaccharide-binding protein is described. 2. The protein was purified from EDTA extracts by a two-step procedure consisting of ion-exchange chromatography on CM-Sephadex and preparative polyacrylamide gel electrophoresis at pH 9.5. The yield of the total purification procedure was around 16%. 3. The resulting protein preparation was homogeneous on the basis of disc gel electrophoresis, dodecylsulfate gel electrophoresis, isoelectric focusing in polyacrylamide gel and immunoelectrophoresis. 4. The isoelectric point of the protein was found to be 10.3 at 4 degrees C. Its molecular weight determined by dodecylsulfate gel electrophoresis is 15000. Its amino acid composition is characterized by the absence of histidine and proline, a low content in tyrosine and high amounts of alanine, lysine, aspartic and glutamic acid residues, or their respective amides. 5. The lipopolysaccharide-protein association was shown to be mainly due to ionic interactions of the basic protein with negatively charged groups (probably phosphate and pyrophosphate groups) of the lipid A moiety. 6. Purified lipopolysaccharide-binding protein is immunogenic in rabbits, thus enabling the preparation of specific antiserum. 7. The protein is located at the surface of Salmonella minnesota Re mutant cells as revealed by antiserum absorption with total bacteria. Ferritin-labelling studies further demonstrated that it is evenly spread over the entire cell surface. 8. Comparative antiserum absorption studies using smooth and rough strains of Salmonella minnesota, Salmonella typhimurium, Escherichia coli, Klebsiella and Shigella revealed the presence of lipopolysaccharide-binding protein (or a serologically cross-reacting antigen) in most of the strains tested. From these results the protein can be considered as a common antigen of Enterobacteriaceae.

Amino Acids↗