Pastuszak I, Kaushal G P, Wall K A, Pan Y T, Sturm A, Elbein A D
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Glycobiology. 1990 Sep;1(1):71-82. doi: 10.1093/glycob/1.1.71.
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.
从绿豆幼苗的可溶性组分和微粒体组分中纯化出两种芳基甘露糖苷酶（分别标记为A和B），达到了均一性。微粒体中的芳基甘露糖苷酶A在天然凝胶和SDS凝胶上与可溶性芳基甘露糖苷酶A表现相同，芳基甘露糖苷酶B也是如此。沉降速度研究表明这两种酶都是均一的，芳基甘露糖苷酶A的分子量为237 kd，而B的分子量为243 kd。芳基甘露糖苷酶A在SDS凝胶上显示出两条主要蛋白带，分子量分别为60 kd和55 kd，还有79 kd、39 kd和35 kd的次要条带。所有这些条带都是N-连接的，因为它们易受内切葡糖胺聚糖酶H的消化作用。此外，至少主要条带可以用针对N-连接植物寡糖木糖部分产生的抗体通过蛋白质免疫印迹法检测到，并且它们也可以在大豆悬浮细胞中用[2-³H]甘露糖进行标记。芳基甘露糖苷酶B显示出三条主要条带，分子量分别为72 kd、55 kd和45 kd，还有42 kd和39 kd的次要条带。除了45 kd和42 kd的条带可能例外，所有这些条带都是糖蛋白。就从高甘露糖寡糖中释放甘露糖而言，芳基甘露糖苷酶A和B表现出略有不同的动力学，但它们对苦马豆素和甘露糖抑素A的抑制同样敏感。针对芳基甘露糖苷酶B产生的多克隆抗体与绿豆幼苗中的芳基甘露糖苷酶A以及大豆细胞中的芳基甘露糖苷酶具有同样良好的交叉反应。然而，针对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的作用效果要差得多。利用抗体检测了在[2-³H]甘露糖中生长的大豆细胞中芳基甘露糖苷酶碳水化合物链的生物合成和结构。用内切葡糖苷酶H处理纯化的酶释放出约50%的放射性，这些标记的寡糖是高甘露糖类型，即主要是Man9GlcNAc。从内切葡糖苷酶H处理中分离出的沉淀蛋白仍含有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。
