Gunda Keilich

1H-NMR-Spektroskopische untersuchungen zur mutarotation der N-acetyl-D-neuraminsäure-ph-abhängigkeit der mutarotationsgeschwindigkeit

Abstract The pD-dependence of the mutarotation of N-acetyl-D-neuraminic acid was studied by 1 H-nmr spectroscopy. The rate constant has a minimum at pD 5.4. At pD 1.3 and pD 11.7 the establishment of the equilibrium of mutarotation was found to be so fast, that it cannot be measured.

Circular dichroism studies on α- and β-ketosides of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives☆

Abstract The circular dichroism spectra of a number of N -acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methyl, ethyl, and allyl α- D -ketosides are characterized by a broad, positive band centered at λ ≈ 195 nm with a slight skew towards the higher wavelengths and weak bands between λ 225 and 255 nm, whereas the methyl β- D -ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy- D - glycero -β- D - galacto -nonulopyranose, its methyl β- D -ketoside, and 5-acetamido-3,5-dideoxy- D - glycero - D - galacto -nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at λ ≈ 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy- D - glycero - D - galacto -nonulopyranosonic acid confirms the β- D configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O -( N -acetyl-α- D -neuraminopyranosyl)-(2→3)- O -β- D -galactopyranosyl-(1→4)- D -glucopyranose resembles that of the methyl, ethyl, and allyl α- D -ketosides 2-4 .

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 β-galactoside-specific lectin,Ricinus communis agglutinin (RCA). Sialidase and β-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 β-galactosidases fromS. 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.

REVERSIBLE IMMOBILIZATION OF VIBRIO CHOLERAE NEURAMINIDASE BY BIOSPECIFIC SORPTION

It has been established that N-(4-nitrophenyl)oxamic acid coupled with 1.6-diami-nohexyl Sepharose 4B is a specific adsorbent for V. cholerae neuraminidase. The enzyme immobilized by non-covalent linkages retains its full activity. Natural and synthetic substrates were hydrolyzed in 50 mM sodium acetate buffer pH 5.0, containing 2 mM calcium ions; however, with none of the substrates (up to 50 umol/ml gel) any enzyme activity was eluted from the adsorbent. The adhesion of neuraminidase is reversible; it may be desorbed by sodium acetate buffer pH 5.0, containing 1.0 M sodium chloride. A simple method for the preparation of asialo-glycoproteins is presented, whereby the immobilized neuraminidase may be used continuously without loosing its activity.