Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.
A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.
Kochibe
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Department of Biology, Faculty of Education, Gunma University, Maebashi, Japan.
Matta
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D000117:Acetylglucosamine; D000596:Amino Acids / Q000032:analysis; D001487:Basidiomycota; D002241:Carbohydrates; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D005944:Glucosamine / Q000031:analogs & derivatives; D006384:Hemagglutination; D007700:Kinetics; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000302:isolation & purification*; D034781:Receptors, N-Acetylglucosamine
D000596:Amino Acids; D002241:Carbohydrates; D011971:Receptors, Immunologic; D034781:Receptors, N-Acetylglucosamine; D005944:Glucosamine; D000117:Acetylglucosamine