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 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 beta-1-3-N-acetylgalactosamine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for alpha-2-6 and alpha-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax flavus (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.
Rogerieux
F
F
INSERM U 139, Hopital, H. Mondor, Créteil, France.
Belaise
M
M
Terzidis-Trabelsi
H
H
Greffard
A
A
Pilatte
Y
Y
Lambré
C R
CR
D002240:Carbohydrate Sequence; D005456:Fluorescent Dyes; D005690:Galactose / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000378:metabolism; D037121:Plant Lectins; D012015:Reference Standards; D054788:Ribosome Inactivating Proteins; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D000509:alpha-Fetoproteins / Q000378:metabolism
D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D012794:Sialic Acids; D000509:alpha-Fetoproteins; D009439:Neuraminidase; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid; D005690:Galactose