Purification and properties of arylmannosidases from mung bean seedlings and soybean cells.
Purification and properties of arylmannosidases from mung bean seedlings and soybean cells.
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DOI：
10.1093/glycob/1.1.71
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发表时间：
1990-09
期刊：
影响因子：
4.3
通讯作者：
I. Pastuszak;G. Kaushal;K. Wall;Y. T. Pan;A. Sturm;A. Elbein
中科院分区：
文献类型：
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作者：
I. Pastuszak;G. Kaushal;K. Wall;Y. T. Pan;A. Sturm;A. Elbein
从绿豆幼苗的可溶性组分和微粒体组分中纯化了两种芳基甘露糖苷酶（标记为A和B）。在天然凝胶和SDS凝胶上，来自微粒体的芳基甘露糖苷酶A与可溶性芳基甘露糖苷酶A相同，芳基甘露糖苷酶B也是如此。沉降速度研究表明，这两种酶是均匀的，芳基甘露糖苷酶A的分子量为237 kd，而B的分子量为243 kd。在SDS凝胶上，甘露糖苷酶A有两条主要蛋白带，分子量分别为60和55 kd，其次为79、39和35 kd。所有这些条带都是N-连锁的，因为它们对内切氨基葡萄糖苷酶H的消化敏感。此外，至少主要的条带可以通过Western印迹与针对N-连接的植物寡糖的木糖部分的抗体检测，并且它们也可以在大豆悬浮细胞中用[2- 3 H]甘露糖标记。芳基甘露糖苷酶B有3条主带，分子量分别为72、55和45 kd，次带为42和39 kd。除45和42 kd条带外，所有这些条带都是糖蛋白。芳基甘露糖醛酸酶A和B在从高甘露糖寡糖释放甘露糖方面表现出稍微不同的动力学，但它们对苦马豆素和甘露糖抑制素A的抑制同样敏感。针对芳基甘露糖苷酶B的多克隆抗体与来自绿豆幼苗的芳基甘露糖苷酶A和来自大豆细胞的芳基甘露糖苷酶交叉反应同样好。然而，抗绿豆芳基甘露糖苷酶A的单克隆抗体对芳基甘露糖苷酶B的效果要差得多。用抗体检测了在[2- 3 H]甘露糖中生长的大豆细胞中芳基甘露糖苷酶糖链的生物合成和结构。用Endo H处理纯化的酶释放约50%的放射性，并且这些标记的寡糖是高甘露糖型的，即主要是Man 9 GlcNAc。从Endo H处理中分离的沉淀蛋白仍含有50%的放射性，并且这存在于可能含有木糖残基的修饰结构中。
Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings. Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B. Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd. Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd. All of these bands were N-linked since they were susceptible to digestion by endoglucosaminidase H. In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with [2-3H]mannose. Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd. With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins. Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A. Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells. However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B. Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in [2-3H]mannose. Treatment of the purified enzyme with Endo H released approximately 50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e. mostly Man9GlcNAc. The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residues.