The action of sialidases on substrates containing O-acetylsialic acids.

O-Acetyl substitution of sialic acids in glycoconjugates reduces the rate of action of sialidases on these substrates. A plasma glycoprotein fraction and an erythrocyte ganglioside containing 4-O-acetylsialic acids were isolated and characterized from equine blood, and a sialyllactose preparation with Neu5,9Ac2 was purified from rat urine. Using the novel substrates II3Neu4Ac5Gc-LacCer and II3Neu5,9Ac2-Lac the influence of individual mono-O-acetylated sialic acids on bacterial and viral sialidases could be clearly shown. This extends and clarifies observations with glycoproteins containing mixtures of mono-, di- and higher O-acetylated sialic acids with substitution at the hydroxyls on carbons 4, 7, 8 and 9. A 4-O-acetyl substitution in sialic acids blocks the action of bacterial sialidases for substrates containing these derivatives, while viral enzymes show low but significant activity, reflected in Km and Vmax values. A small reduction in bacterial sialidase activity was observed for II3Neu5,9Ac2-Lac relative to II3Neu5Ac-Lac in agreement with kinetic analysis. Newcastle disease virus sialidase showed a 50% reduction in hydrolysis rate for the 9-O-acetylated substrate and ten-fold reductions of both Km and Vmax values.

Sialidase activity in the sera of patients and rabbits with clostridial myonecrosis.

The origin and nature of gas gangrene can be diagnosed exactly only by time-consuming bacteriological tests. In order to improve the diagnostic procedures, rabbits were infected with strains of Clostridium perfringens, Clostridium septicum or Clostridium sordellii. Sialidase activity was found to increase rapidly in serum; elevated creatine kinase activities were observed, too. High sialidase concentrations were found in sera (up to 1.6 mU/ml) and in tissues of wounded regions (up to 110 mU/g) of patients diagnosed to be infected with C. perfringens. By inhibition of enzyme activity with antibodies specific for the sialidase from this Clostridium species, it was possible to identify the clostridial origin of the sialidase activities. In the same material from other patients supposed to suffer from gas gangrene, but where no Clostridia could be detected, significant sialidase activity was not found. Thus, sialidase may be a useful tool for the diagnosis of myonecrosis due to clostridial infection.

New chromatographic system for the rapid analysis and preparation of colostrum sialyloligosaccharides.

A new thin-layer chromatographic system on silica gel for the separation of sialyloligosaccharides is described. Calibration of the system with standard milk and colostrum sialyloligosaccharides is presented. The use of the system in monitoring different oligosaccharides is demonstrated for the purification of bovine colostrum sialyllactose isomers and a commercial sialyllactose product, and is discussed with respect to other biological fluids. The large-scale preparation of pure sialyllactose isomers from bovine colostrum is achieved using an improved ion-exchange separation on Dowex 1-X2 (less than 400 mesh) employing isomolar elution at 20 mM for monosialyloligosaccharides and 200 mM for disialyllactose. The purification of four major monosialyltrisaccharides, the 2-3 and 2-6 isomers of N-acetylneuraminyllactose, N-glycolylneuraminyl2-3lactose and N-acetylneuraminyl2-6-N-acetyllactosamine, and the disialyltetrasaccharide di-N-acetylneuraminyllactose is reported. The detection and partial purification of three new minor monosialyloligosaccharides is described.