Kaushal G P, Szumilo T, Pastuszak I, Elbein A D
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Biochemistry. 1990 Feb 27;29(8):2168-76. doi: 10.1021/bi00460a030.
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.
通过使用包括DEAE - 纤维素和羟基磷灰石色谱、凝胶过滤、凝集素亲和色谱以及制备性凝胶电泳等多种技术组合，从绿豆幼苗中纯化出甘露糖苷酶II至表观均一性。从GlcNAc[3H]Man5GlcNAc释放放射性甘露糖与时间和纯化蛋白的蛋白质浓度呈线性关系，不显示任何金属离子需求，最适pH为6.0。纯化后的酶在SDS凝胶上呈现单一条带，与Mr 125K标准品迁移情况相同。该酶对GlcNAcMan5GlcNAc具有很高活性，但对Man5GlcNAc、Man9GlcNAc、Glc3Man9GlcNAc或其他高甘露糖寡糖无活性。它对Man3GlcNAc显示出轻微活性。通过凝胶过滤分离出酶与GlcNAcMan5GlcNAc反应的首个产物，即GlcNAcMan4GlcNAc，并对其进行内切葡糖胺酶H消化，以确定去除了哪个甘露糖残基。这种GlcNAcMan4GlcNAc约60%易受内切葡糖苷酶H作用，表明甘露糖苷酶II优先去除α1,6连接的甘露糖，但40%的情况下首先去除α1,3连接的甘露糖。反应的最终产物GlcNAcMan3GlcNAc通过凝胶过滤和各种酶促消化进行表征。甘露糖苷酶II受到苦马豆素的强烈抑制，受到1,4 - 二脱氧 - 1,4 - 亚氨基 - D - 甘露糖醇的抑制较弱。它不受脱氧野尻霉素的抑制。
