Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.
Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.
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发表时间：
1989-01
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通讯作者：
N. Kochibe;K. L. Matta
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作者：
N. Kochibe;K. L. Matta
采用甲壳素亲和层析和离子交换层析从绒毛Psathyrella velutina子实体中分离纯化了一种凝集素。绒毛青霉凝集素（PVL）在缓冲盐水中倾向于不可逆地聚集，但发现向凝集素溶液中添加甘油（10%，v/v）可以防止聚集体形成。假设PVL作为Mr = 40，000的多肽的单体存在，如通过凝胶过滤和在十二烷基硫酸钠存在下通过凝胶电泳所测定的。PVL对N-乙酰葡糖胺（GlcNAc）具有特异性。通过平衡透析测定，每个多肽分子有4个结合位点，显示与该糖的平均内在缔合常数K 0 = 6.4 x 10（3）M-1。凝集素的结合特异性进行了研究，通过血凝抑制试验，并通过亲和素-生物素介导的酶免疫试验，使用各种含GlcNAc的酶。结果表明，甲基N-乙酰基β-氨基葡糖苷是比相应的α-端基异构体稍好的抑制剂。PVL与带有1-6或1-3连接的非还原性末端β-GlcNAc的寡糖结合良好，但与具有1-4连接的寡糖结合较差，如N-乙酰化壳寡糖。当其在C-6位置被取代时，其还结合亚末端GlcNAc部分，但当在C-3或C-4位置被取代时，其不与该部分相互作用。因此，这些结果表明PVL在其结合特异性方面与其他高等植物的GlcNAc结合凝集素完全不同，因为它们优先结合1- 4键的β-GlcNAc，并且它们对几丁质寡糖具有高亲和力。
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.