Purification and properties of alpha-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells.
Purification and properties of alpha-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells.
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杆状病毒感染的草地贪夜蛾 (IPLB-SF-21AE) 细胞高尔基样膜中 α-甘露糖苷酶 II 的纯化和特性。
DOI：
10.1042/bj3240951
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Bretthauer,RK
中科院分区：
文献类型：
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作者：
Ren,J;Castellino,FJ;Bretthauer,RK
在杆状病毒感染的果蝇细胞中发现了类α甘露糖苷酶II活性。采用DEAE-纤维素、羟基磷灰石、刀豆蛋白A-琼脂糖凝胶和凝胶过滤层析等步骤，从高尔基体膜中纯化得到明显的均一酶。该纯化蛋白的相对分子质量约为100%。还原条件下约120 kDa的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳。在非还原条件下为240 kDa，表明该酶为二硫键连接的二聚体。该酶的底物为GlcNAc-Man5-GlcNAc-GlcNAc(未还原和还原)和对硝基苯基α-d-甘露糖苷。低聚糖底物通过中间体GlcNAc-Man4-GlcNAc-GlcNAc转化为GlcNAc-Man3-GlcNAc-GlcNAc。分离得到的中间低聚糖经内切糖苷酶H处理后转化为GlcNAc-Man4-GlcNAc。这表明它在α-1，6-甘露糖臂上含有α-1，3-甘露糖残基，并表明α-1，6-甘露糖臂上的甘露糖残基优先被甘露糖苷酶降解。缺少β-1，2-甘露糖臂上的α-1，2-GlcNAc残基的低聚糖(Man5-GlcNAc-GlcNAc)在该酶的存在下不能被水解。金属离子在任何底物上都不需要酶活性，但Cu2+对酶有很强的抑制作用。低浓度的苦马豆素对该酶有抑制作用，而高浓度的1-脱氧甘露糖苷才能抑制该酶的活性。所有这些性质都是来自其他真核组织的甘露糖苷酶II的特征。鳞翅目昆虫细胞中存在甘露糖苷酶II可以使N-连接的糖蛋白进入复杂的加工反应途径或进入末端的Man3-GlcNAc-GlcNAc途径。
An α-mannosidase II-like activity was identified in baculovirus-infectedSpodoptera frugiperda(IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) andp-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.