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R Brossmer

Publications and source records attributed to R Brossmer.

At least 73 records · Page 4Linked to original sources
PubMed1989

[Cleavage and synthesis of sialic acids with aldolase. NMR studies of stereochemistry, kinetics, and mechanisms].

1H-NMR spectroscopy was used to study cleavage and synthesis of N-acetyl- and N-glycoloyl-D-neuraminic acid by Clostridium perfringens aldolase. Whereas the alpha-anomers of Neu5Ac and Neu5Gc serve as substrate in the cleavage reaction, alpha-ManNAc and alpha-ManNGc are its primary products. The same alpha-anomers are needed by the aldolase for the synthesis of Neu5Ac and Neu5Gc. During the enzyme reaction in D2O both H-atoms at C-3 of Neu5Ac are exchanged by deuterium, H-3e reacting faster than H-3a. Rate constants and concentrations at equilibrium of reactants are temperature- and pH-dependent: The amount of Neu5Ac in equilibrium increases with decreasing temperature and increasing pH-value. Based on these results a mechanism of aldolase action is discussed.

Clostridium perfringens↗
PubMed1988

Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins.

Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5-acetamido-9-(3-fluoresceinylthio-ureido)-3,5,9-trideoxy-2-non ulosonic acid (9-fluoresceinyl-NeuAc) was synthesized which has an intact carbon chain. a) Despite the space-filling substituent at C-9, the fluorescent NeuAc analogue was activated to the corresponding CMP-glycoside by CMP sialic acid synthase from bovine brain. Whereas the Km value of the synthase was little affected by the modification (Km = 2.1 mM, for NeuAc Km = 1.4 mM), the V value decreased to 7.5%. b) CMP-9-fluoresceinyl-NeuAc was synthesized on a preparative scale (17% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra. c) 9-Fluoresceinyl-NeuAc was transferred onto asialo-alpha 1-acid glycoprotein by both Gal beta 1, 4GlcNAc alpha 2, 6sialyltransferase and Gal beta 1,4(3)GlcNAc alpha 2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc alpha 2,6-sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio cholerae sialidase. d) Initial rate studies indicated a low Km value of Gal beta-1,4GlcNAc alpha 2,6sialyltransferase, and GalNAc alpha 2,6sialyltransferase (specific for O-linked oligosaccharide chains) for CMP-9-fluoresceinyl-NeuAc.

Animals↗
PubMed1988

Purification of alpha 2,6-sialyltransferase from rat liver by dye chromatography.

We describe a simple three-step purification for Gal-beta 1,4-GlcNAc-alpha 2,6-sialyltransferase (EC 2.4.99.1) from rat liver which uses chromatography on Cibacron Blue F3GA and f.p.l.c. It gives a highly purified (11,000-fold) enzyme in 19% yield, which is free of other sialyltransferases, CMP-NeuAc hydrolase, sialidases and proteinases.

Animals↗
PubMed1988

Interaction of N-acetyl-4-epi-D-neuraminic acid with key enzymes of sialic acid metabolism.

In spite of the axially orientated hydroxy group at C-4, the benzyl alpha-glycoside of N-acetyl-4-epi-D-neuraminic acid (4-epi-NeuAc) is a substrate for sialidases from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, although to an extent which differs depending on the enzyme. Surprisingly, V. cholerae sialidase is by far the slowest acting enzyme; this is in contrast to its usual behavior. Fowl plague virus sialidase and bovine testis sialidase also cleave this glycoside slowly. 4-Epi-NeuAc is not a substrate for N-acetylneuraminic acid aldolase from C. perfringens but reversibly inhibits the enzyme with a Ki = 2.3 mM. The N-acetylneuraminic acid analogue is not converted to the corresponding CMP-glycoside by CMP-sialic acid synthase from bovine brain; however, it is an effective reversible inhibitor of the enzyme. The kinetic properties were analyzed with an assay system at pH 9 as well as an assay system at pH 7.5. The results from Dixon and Hanes plots did not agree. Therefore, no conclusions about the mechanism of the inhibition could be reached. This is the first reported sialic acid analogue which can act as an inhibitor of CMP-sialic acid synthase.

Animals↗
PubMed1988

Inhibition of N-acetylneuraminate lyase by N-acetyl-4-oxo-D-neuraminic acid.

We show that the 4-oxo analogue of N-acetyl-D-neuraminic acid strongly inhibits N-acetylneuraminate lyase (NeuAc aldolase, EC 4.1.3.3) from Clostridum perfringens (Ki = 0.025 mM) and Escherichia coli (Ki = 0.15 mM). In each case the inhibition was competitive. N-Acetyl-D-neuraminic acid; N-Acetylneuraminate lyase; N-Acetyl-D-neuraminic acid analog; 5-Acetamido-3,5-dideoxy-beta-D-manno-non-2,4-diulosonic acid; 2-Deoxy-2,3-didehydro-N-acetyl-4-oxo-neuraminic acid; Competitive inhibitor.

Binding, Competitive↗
PubMed1988

Complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein and its homology to histocompatibility antigens.

In the present study the complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz 1H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approximately equal to 41,000 based on physicochemical measurements. The predicted secondary structure appeared to be comprised of 23% alpha-helix, 27% beta-sheet, and 22% beta-turns. The three N-glycans were found to be located in beta-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-alpha 2-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the alpha chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-alpha 2-glycoprotein appears to be a truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.

Amino Acid Sequence↗
PubMed1988

Removal of N-linked carbohydrates decreases the infectivity of herpes simplex virus type 1.

Purified preparations of herpes simplex virus type 1 Angelotti were digested with the exoglycosidases sialidase, beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-mannosidase, and with the endoglycosidases Endo-H and Endo-F. It was found that treatment of virions with Endo-F specifically decreased viral infectivity by a factor of 10. This reduction in titre was not associated with any measurable differences in virus adsorption, suggesting a role of N-linked complex type oligosaccharide chains in penetration. In contrast, a reduction in titre observed upon digestion of virions with exoglycosidases could be attributed to a proteolytic contamination in these enzyme preparations. Treatment of virions with Endo-H, demonstrated to be free of proteolytic contamination, did not reduce viral infectivity. Analysis of endoglycosidase-digested virions by monospecific antibodies and immunoblotting revealed a susceptibility of all four major glycoproteins (gC, gB, gE and gD) to Endo-F, but only gB was susceptible to Endo-H treatment. In contrast, of all the exoglycosidases used only sialidase was found to be active towards native viral glycoproteins. Upon analysis of endoglycosidase-digested virions we could not find any evidence for proteolysis, degradation or altered protein composition of viral envelopes. In contrast, vigorous inhibition of glycoprotein glycosylation by tunicamycin led to the formation of physically intact virions almost completely lacking all major glycoproteins. These data show that digestion of intact virions with glycosidases allows an analysis of the functional relevance of carbohydrate residues without any obvious alterations in the virion glycoprotein composition.

Adsorption↗
PubMed1987

Activation and transfer of novel synthetic 9-substituted sialic acids.

In this report several NeuAc analogues differently modified at position C-9 were tested as substrates for CMP sialic acid synthase from bovine brain: the hydroxy group at C-9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group. The synthase was partially purified by chromatography on CDP-hexanolamine--Sepharose. CMP-glycosides synthesized were measured by analytical HPLC at 275 nm. Each NeuAc analogue was activated to the respective CMP-glycoside: Km-values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C-9. CMP-glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500-MHz 1H-NMR data of CMP-9-amino-NeuAc and CMP-9-acetamido-NeuAc were obtained. Each CMP-activated NeuAc analogue was a suitable donor substrate for Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non-labelled NeuAc analogues can be incorporated into glycoprotein with high yield.

Animals↗
PubMed1987

Structural basis for altered soybean agglutinin lectin binding between a murine metastatic lymphoma and an adhesive low malignant variant.

By selection for plastic adhesiveness we have previously established a variant tumor line (ESb-MP) from the metastatic murine lymphoma ESb. In contrast to the parental line, the adhesion variant is significantly decreased in malignancy and is altered in the capacity to bind soybean agglutinin (SBA) lectin. Here we show biochemically that the major SBA-binding cell-surface component of ESb-MP cells is the T200 glycoprotein. In ESb cells, T200 antigens bind SBA only after sialidase treatment. Enzymatic studies suggested that glycans detected by the lectin with or without sialidase treatment are different. Inhibition of N-glycosylation by tunicamycin and biosynthetic labeling revealed two T200 chains for ESb-MP cells that were larger in size than the single chain detected in ESb cells. Studies on the biosynthesis revealed that ESb-MP cells expressed two precursor chains for T200 whereas ESb cells displayed only one. There was no size difference detectable in the mature T200 molecules of ESb and ESb-MP cells. Our data suggest that the molecules differ in expression of O-linked glycans that can be recognized by SBA. Additional O-linked sugars on ESb-MP T200 molecules seem to be expressed in particular after trimming of the second T200 precursor chain.

Animals↗
PubMed1987

Recognition of monovalent sialosides by influenza virus H3 hemagglutinin.

This report presents a procedure for evaluating the relative affinities of simple sialoside receptor determinants for their interaction with the receptor binding pocket of the influenza virus hemagglutinin. The virus examined was A/Memphis/102/72, previously shown to exhibit preferential binding specificity for enzymatically modified erythrocytes containing the NeuAc alpha 2,6Gal linkage. Sialosides examined included those with structures representative of terminal sequences found on carbohydrate groups of naturally occurring glycoproteins and glycolipids. Sialosides with the NeuAc alpha 2,6Gal and NeuAc alpha 2,6GlcNAc linkage were 5-30 times more potent inhibitors than those with the NeuAc alpha 2,3Gal linkage. The results provide evidence that the previously reported specificity of the A/Memphis/102/72 hemagglutinin for the NeuAc alpha 2,6Gal sequence on cell surface receptors was due to differential affinity of the receptor binding pocket for sialoside sequences, apart from contributions due to the protein or lipid portions of the cell surface receptors. A number of synthetic sialic acid glycosides were also evaluated as inhibitors of viral attachment. The differential inhibition observed with these receptor analogs may provide clues to the detailed molecular interaction of sialosides with the hemagglutinin binding pocket and aid in the design of small molecular weight inhibitors of viral adsorption to cells.

Binding, Competitive↗
PubMed1986

Variation and adaptation of Pseudomonas aeruginosa toxicity to HeLa cells and fibroblasts.

The toxic components of supernatants from Pseudomonas aeruginosa cultures directed against HeLa cells and Staphylococcus aureus were evaluated with the aim of discovering interactions. Supernatants of eight different strains of P. aeruginosa were assayed for cytotoxic activity. All were active against HeLa cells; seven were toxic for S. aureus. On repeated suspension of P. aeruginosa in 0.9% sodium chloride solution, a shift from HeLa cell toxicity to staphylococcal lytic activity occurred along with a change of toxic activity from a high (50,000 +/- 5,000) to a low (8,000 +/- 400) molecular weight (MW) range on gel filtration. Addition of protein to the minimal medium of cultures producing material toxic only for S. aureus reactivated the generation of HeLa cell-toxic material. Cultivation of P. aeruginosa in the presence of HeLa cells and a chloramphenicol supplement produced suppression of the generation of material toxic for S. aureus but facilitated that of HeLa-toxic material of high MW. Adaptation of toxicity against fibroblasts developed only on cocultivation of P. aeruginosa together with S. aureus and in the presence of fibroblasts. Under these conditions a strong lytic activity for S. aureus appeared, even in the presence of chloramphenicol. Chloramphenicol caused the material toxic for fibroblasts to elute at a low MW well separated from that toxic for HeLa cells. In contrast to the high-MW toxic substances, the low-MW material did not induce antibodies after injection into rabbits. This may explain failures of vaccination against P. aeruginosa infection and of serum therapy of homologous sepsis in humans.

Bacterial Toxins↗
PubMed1985

A comparison of established human lymphoma lines by flow cytometry: quantitation of Ricinus communis agglutinin binding and the effect of specific glycosidases.

Two established cell lines of human B-cell lymphomas derived from Burkitt lymphomas and their Epstein-Barr virus-transformed counterparts were analyzed with respect to their ability to bind the beta-galactoside-specific lectin Ricinus communis agglutinin (RCA). Native and sialidase- as well as sialidase-beta-galactosidase-treated cells were compared. The method for the quantitative determination of average numbers of binding sites and of apparent affinity constants was flow cytometry with fluorescence-labeled lectin. Although with native cells there was no significant deviation of the values for virus-transformed cells from those for the parent cells, some differences could be detected after glycosidase treatment. The general procedure of the combined application of specific glycosidases and the quantitation of sugar-specific lectin binding is recommended as a general strategy for the differentiation of cells with known or putative differences in biological functions.

Binding Sites↗
PubMed1984

The effect of specific glycosidases on Ricinus communis agglutinin binding to cell surfaces of two tumor sublines. A comparative flow-cytometric study.

The effects of the sequential application of specific glycosidases on surfaces of living mammalian cells were studied with respect to their ability to bind the beta-galactoside-specific lectin, Ricinus communis agglutinin (RCA). Sialidase and beta-galactosidases from different sources were tested for their actions on two strains of mouse lymphoma cells differing markedly in their metastatic potential. Binding studies were performed by quantitative flow cytometry with fluorescent RCA, and numbers of specific binding sites and equilibrium association constants for the lectin on living cells were determined before and after the various enzyme treatments. Although the number of binding sites for native and sialidase-treated cells were almost identical for both cell strains, differences in the apparent affinity constants could be detected. Differences between the two strains became even more pronounced, also with respect to the number of binding sites, after treatment with beta-galactosidases from S. pneumoniae and from bovine testis. It is suggested that such combined strategies provide valuable tools for the differentiation of surface carbohydrate moieties on intact living cells, especially for comparative purposes.

Animals↗