Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland.
Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland.
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参与牛乳腺 N 连接糖蛋白加工的葡萄糖苷酶 I 的纯化和表征。
DOI：
10.1042/bj2470555
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Vijay,IK
中科院分区：
文献类型：
--
作者：
Shailubhai,K;Pratta,MA;Vijay,IK
糖苷酶I是第一种参与N-连锁糖蛋白翻译后加工的酶，从泌乳牛乳腺组织中纯化得到均一的糖苷酶。用Triton X-100和Lubrol PX对微粒体组分进行差异性处理，提取酶。以N-5-羧基戊基脱氧诺吉霉素为配基，经亲和层析和DEAE-SepharoseCL-6B柱层析。纯化的葡萄糖苷酶I经Sephacryl S-300凝胶过滤，相对分子质量为320-330 kDa。还原条件下的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法显示约一条单一条带。85 kDa，表明该酶可能是一种四聚体蛋白。几个标准，包括最适的pH为6.6-7.0，对Glc3Man9GlcNAc2的特定水解作用，以释放末端α-1，2连接的葡萄糖残基，以及对处理葡萄糖苷酶II的生物底物Glc1Man9GlcNAc2和Glc2Man9GlcNAc2完全缺乏活性，以及4-甲基伞形花环基α-D-吡喃葡萄糖苷显示了纯化酶的非溶酶体来源和加工特有的作用。该酶的活性不需要任何金属离子。Hg2+、Ag+和Cu2+是该酶的有效抑制剂；这种抑制可以通过加入过量的二硫苏糖醇来逆转。在所测试的糖类中，曲二糖(Glcα1-2Glc)对酶有抑制作用。免疫印迹分析和蛋白A-琼脂糖凝胶免疫吸附实验表明，兔抗该酶的多克隆抗体对葡萄糖苷酶I具有特异性。在来自牛、豚鼠、大鼠和小鼠的肝、乳腺和心脏的溶解微粒体制剂中，抗葡萄糖苷酶I抗体与类似的抗原发生交叉反应。
Glucosidase I, the first enzyme involved in the post-translational processing of N-linked glycoproteins, was purified to homogeneity from the lactating bovine mammary tissue. The enzyme was extracted by differential treatment of the microsomal fraction with Triton X-100 and Lubrol PX. The solubilized enzyme was subjected to affinity chromatography on Affi-Gel 102 with N-5-carboxypentyldeoxynojirimycin as ligand and DEAE-Sepharose CL-6B chromatography. Purified glucosidase I shows a molecular mass of 320-330 kDa by gel filtration on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis under reducing conditions indicates a single band of approx. 85 kDa, indicating that the native enzyme is probably a tetrameric protein. Several criteria, including pH optimum of 6.6-7.0, specific hydrolytic action towards Glc3Man9GlcNAc2, to release the terminally alpha-1,2-linked glucosyl residue, and total lack of activity towards Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 saccharides, which are the biological substrates for processing glucosidase II, and 4-methylumbelliferyl alpha-D-glucopyranoside show the non-lysosomal origin and the processing-specific role of the purified enzyme. The enzyme does not require any metal ions for its activity. Hg2+, Ag+ and Cu2+ are potent inhibitors of the enzyme; this inhibition can be reversed by adding an excess of dithiothreitol. Among the saccharides tested, kojibiose (Glc alpha 1----2Glc) was inhibitory to the enzyme. Polyclonal antibodies raised against the enzyme in rabbit were found to be specific for glucosidase I, as revealed by Western-blot analysis and by immunoadsorption with Protein A-Sepharose. Anti-(glucosidase I) antibodies were cross-reactive towards a similar antigen in solubilized microsomal preparations from liver, mammary gland and heart from the bovine, guinea pig, rat and mouse.