Purification to homogeneity and properties of mannosidase II from mung bean seedlings.
Purification to homogeneity and properties of mannosidase II from mung bean seedlings.
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DOI：
10.1021/bi00460a030
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发表时间：
1990-02
期刊：
影响因子：
2.9
通讯作者：
G. Kaushal;T. Szumiło;I. Pastuszak;A. Elbein
中科院分区：
文献类型：
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作者：
G. Kaushal;T. Szumiło;I. Pastuszak;A. Elbein
采用deae -纤维素和羟基磷灰石层析、凝胶过滤、凝集素亲和层析和制备凝胶电泳等技术，从绿豆幼苗中纯化甘露糖苷酶II，使其具有明显的均匀性。GlcNAc[3H]Man5GlcNAc对放射性甘露糖的释放与纯化蛋白的时间和蛋白浓度呈线性关系，不需要金属离子，最适pH为6.0。纯化后的酶在SDS凝胶上显示为单条带，用Mr 125K标准迁移。该酶对GlcNAcMan5GlcNAc很有活性，但对Man5GlcNAc、Man9GlcNAc、Glc3Man9GlcNAc及其他高甘露糖低聚糖无活性。它确实显示出对Man3GlcNAc的轻微活性。酶与GlcNAcMan5GlcNAc反应的第一个产物，即GlcNAcMan4GlcNAc，通过凝胶过滤分离出来，用内切葡萄糖氨基酶H消化，确定去除了哪些甘露糖残留物。该GlcNAcMan4GlcNAc对Endo H的敏感性约为60%，表明甘露糖苷酶II倾向于首先去除α 1,6-连接的甘露糖，但40%的情况下首先去除α 1,3-连接的甘露糖。该反应的最终产物GlcNAcMan3GlcNAc通过凝胶过滤和各种酶解进行了表征。甘露糖苷酶II受到苦马豆素的强烈抑制，而1,4-二脱氧-1,4-亚氨基-d -甘露醇的抑制作用较弱。脱氧甘露诺吉里霉素对其无抑制作用。
Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.