Determination of the Sialic Acid Linkage Specificity of Sialidases Using Lectins in a Solid Phase Assay

A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich meth...

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Bibliographic Details
Published in:Analytical biochemistry 1993-06, Vol.211 (2), p.200-204
Main Authors: Rogerieux, F., Belaise, M., Terzidistrabelsi, H., Greffard, A., Pilatte, Y., Lambre, C.R.
Format: Article
Language:English
Subjects:
alpha-Fetoproteins - metabolism
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Carbohydrate Sequence
Enzymes and enzyme inhibitors
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Galactose - metabolism
Hydrolases
Lectins - metabolism
Molecular Sequence Data
N-Acetylneuraminic Acid
Neuraminidase - chemistry
Neuraminidase - metabolism
Orthomyxoviridae - metabolism
Plant Lectins
Reference Standards
Ribosome Inactivating Proteins
Sialic Acids - chemistry
Sialic Acids - metabolism
Substrate Specificity
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Summary:A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich method which measured the binding of different lectins to fetuin. The lectins used were peanut agglutinin (PNA) from Arachis hypogaea, which binds specifically the galactose β-1-3- N-acetylgalactos-amine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for α-2-6 and α-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax fiavus (LFA) that is specific for N-acetyl and N-glycolyl neuraminic acids. Increased PNA and decreased LFA, SNA, and MAA lectin binding correlated with sialidase-induced desialylation of the substrate. In this report, the assay was used to determine the activities and specificities of influenza, Vibrio cholerae, and Arthrobacter ureafaciens sialidases.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1993.1257