Roland Schauer

Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.

Identification of O-acetylated N-acylneuraminic acids by mass spectrometry

A number of O-acetylated N-acylneuraminic acids, isolated from submandibular glands of cow and horse and from horse erythrocytes, have been characterized by mass spectrometry. On the basis of the typical fragmentation patterns of the pertrimethylsilyl derivatives of the methyl esters of the compounds, they were identified as 4-O-acetyl-, 9-O-acetyl-, 4,9-di-O-acetyl-, and 7,9-di-O-acetyl N-acetylneuraminic acid, and 4-O-acetyl-and 9-O-acetyl-N-glycolylneuraminic acid.

Gas-liquid chromatography of N- and O-acylated neuraminic acids

Abstract The qualitative and quantitative analysis by gas-liquid chromatography of natural and synthetic sialic acids with special reference to O -acylated sialic acids is described. Sialic acids are derivatized by N -trimethylsilimidazol. All hydroxyl groups including the carboxyl group are trimethylsilylated as was shown by mass spectrometry. No elimination of the O -acyl groups occurs during the silylation reaction. The TMS-derivatives of the sialic acids are stable in the refrigerator. The TMS-sialic acids including the isomeric N -acyl- O -acylneuraminic acids can be separated from each other on OV-17 or OV-22. Single peaks having individual retention indices from the following sialic acids were obtained: N -acetylneuraminic acid, N -glycolylneuraminic acid, N -fluoroacetylneuraminic acid and N -chloroacetylneuraminic acid; neuraminic acid-β-methylglycoside or its methylester and N -bromoacetylneuraminic acid-β-methylglycoside; N -acetyl-4- O -acetylneuraminic acid, N -acetyl-7- O -acetylneuraminic acid and N -acetyl-9- O -acetylneuraminic acid; N -acetyl-7,9-di- O -acetylneuraminic acid; N -acetyl-4- O -glycolylneuraminic acid, N -acetyl-9(?)- O -glycolylneuraminic acid and N -acetyl-7(?)- O -glycolylneuraminic acid; N -glycolyl-4- O -acetylneuraminic acid, N -glycolyl-9- O -acetylneuraminic acid and N -glycolyl-7(?)- O -acetylneuraminic acid. On OV-1 N -acetylneuraminic acid and N -glycolylneuraminic acid can be separated from each other and from N -acyl- O -acylneuraminic acids: however, the O -acylated sialic acids elute essentially together from this material. The relative detector responses for the different sialic acids vary, as was shown for N -acetylneuraminic acid, N -glycolylneuraminic acid and N -acetyl-9- O -acetylneuraminic acid. 35% of the radioactivity from radioactive N -acetylneuraminic acid and N -glycolylneuraminic acid eluted correspondingly with the sialic acid peaks detected by the FID. Gas-liquid chromatography enables a sensitive qualitative and quantitative analysis of different sialic acids occurring in biological materials, produced during chemical synthesis or in enzymic assays.

Physical and chemical properties of the mucin secreted by Drosera capensis

Abstract The mucin droplets secreted by the leaves of Drosera capensis consist of a 4 % aq. solution of an acidic polysaccharide containing xylose, mannose,

An improved method for the synthesis of 14C-labelled or 3H-labelled N-acetylneuraminic acid

Abstract 1. 1. Neuraminic acid-β-methylglycoside was prepared from the products obtained by methanolysis of edible bird nest substance (yield, 80%). The methylglycoside was converted to the corresponding methylester hydrochloride by methanolHCl treatment and crystallized from dioxan-light petroleum (yield, 50%). Its properties including mass spectrometry are described. 2. 2. A simple procedure for snythesis of N-[1-14C] acetylneuraminic acid and N-[ 3 H ] acetylneuraminic acid (yields, 75–85%) with high specific radioactivities from the methylester from neuraminic acid-β-methylglycoside and 1-14C- or 3H-labelled acetic anhydride in absolute methanol is described. By this acetylation in water-free solution up to 85% of the theoretically expected amount of radioactivity was found in the N-acetyl groups of neuraminic acid.

Demonstration of Glycoprotein- and Glycolipid-Specific Neuraminidases in Horse Liver

Publisher Summary This chapter discusses the demonstration of glycoprotein and glycolipid-specific neuraminidases in horse liver and other vertebrate species. It also presents the basic criteria to distinguish between different neuraminidases in the same tissue. In an experiment described in the chapter, glycoproteins and gangliosides were labeled in the C-6 side-chain of the neuraminic acid residue by periodate oxidation and borotritide reduction. T.l.c of radioactive labeled compound had shown the specificity of the neuraminidases with collocalia glycopeptides. The enzyme activity was stable under incubation conditions up to 4 h and increased linearly up to 12 mg of protein/ml. This chapter also presents a comparison of neuraminidases activities toward the two substrates and the influence of N-acety1-2, 3-dehydro-2-deoxy-neuraminic acid on neuraminidase activity with the help of graphs.

Abstracts of Free Communications

The surface mucous cells of gastric fundus secrete mucous glycoprotein which covers the inner surface of the stomach. Subcellular fractionation of gastric scrapings in sucrose density gradients yielded a microsomal fraction of 1.12 g/ml buoyant density density, enriched in glycosyl-transferases for galactose, fucose and acetyl-galactosamine, when tested with properly modified glycoproteins as exogenous acceptor molecules. This fraction was devoided of Na+, K+-activated ATPase and of thiamine-pyrophosphatase (TPPase). Electronmicroscopic examination revealed some Golgi-like structures. In the absence of exogenous acceptors the sugar moieties appeared to be transferred to endogenous acceptor molecules. Native glycoprotein, isolated from the gastric mucus layer, did not accept glycosyl-moieties, when added to the fraction. The enzyme involved in endogenous sulphate incorporation appeared to be present in a less dense fraction, enriched in TPPase activity too. The latter enzyme has been demonstrated cytochemically in the mature Golgi cisternae of gastric epithelial cells (J.J. Geuze, unpublished results).

Comparison of Particulate Neuraminidases from Human Heart and Brain

Publisher Summary Mammalian neuraminidases and gangliosides have been reported to occur predominantly in the brain. Gangliosides have been discussed repeatedly with respect to membrane excitation in the nervous system, and neuraminidases may be involved in the regulation of this process. This chapter describes an experiment to examine the possible occurrence of one single neuraminidase in tissues containing excitable membranes, such as human heart and brain tissues. In the experiment, to obtain a radioactive substrate for a highly sensitive neuraminidase assay, the ganglioside GDla was oxidized by periodate and reduced with tritiated borohydride. The results of the experiment suggest strong intrinsic similarities between the particulate human heart and brain neuraminidases.

Isolation and Properties of Acylneuraminate Cytidylyltransferase from Frog Liver

Publisher Summary Frog liver is a rich source for acylneuraminate cytidylyltransferase. This chapter discusses an experiment to study the isolation and properties of enzyme acylneuraminate cytidylyltransferase from frog liver and to assess its value for a ready synthesis of the CMP-glycoside of N-acetylneuraminic acid. It also describes the results from the individual purification of enzyme. The high-molecular weight of the cytidylyltransferase is remarkable. The enzyme undergoes the process of electrophoresis. The inhibition of the enzyme by heavy metal ions and iodoacetamide points to the possible involvement of SH groups in the enzyme reaction. The enzyme is stable in the frozen state, each cycle of freezing and thawing leading to an activity decrease of 15%. It is concluded through experiment that CMP-W-acetylneuraminic acid can easily be synthesized in quantitative yield with the aid of frog liver cytidylyltransferase enriched by chromatography on DEAE-cellulose.