PURIFICATION BY AFFINITY-CHROMATOGRAPHY OF GLUCOSIDASE-I, AN ENDOPLASMIC-RETICULUM HYDROLASE INVOLVED IN THE PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES
PURIFICATION BY AFFINITY-CHROMATOGRAPHY OF GLUCOSIDASE-I, AN ENDOPLASMIC-RETICULUM HYDROLASE INVOLVED IN THE PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES
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DOI：
10.1111/j.1432-1033.1984.tb08253.x
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
BAUSE, E
中科院分区：
文献类型：
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作者：
HETTKAMP, H;LEGLER, G;BAUSE, E
用不同浓度的非离子洗涤剂和盐，从粗小牛肝微粒体中溶解了修剪葡萄糖苷酶I和II，并通过部分提取程序进行了部分浓缩。这两种酶的最适pH均接近6.2，这与溶酶体来源的作用于对-硝基苯苷的水解酶不同，在较酸性的pH条件下，作用速率最高。1-脱氧诺吉霉素对葡萄糖苷酶I、Ⅱ和非特异性α-葡萄糖苷酶(S)均有抑制作用，其半数抑制浓度分别为3微米、20微米、12微米。10脱氧诺吉霉素的N-烷基化大大增强了这些酶之间的区别，并形成了亲和配体的设计基础。以N-羧基戊基-1-脱氧诺吉霉素为配基，经AH-Sepharose4B亲和层析，纯化得到均一的葡萄糖苷酶I。纯化的酶经十二烷基硫酸钠凝胶电泳法测定，亚基分子质量约为85 kDa[kDa]。用凝胶层析法测定该天然酶的相对分子质量为apprxeq。320-350 kDa，指亚基与四聚体的缔合。葡萄糖苷酶I在4℃保存时相当稳定。在洗涤剂存在下(t1/2[半衰期].apprxeq.20天)，并显示出对末端(α)的高度特异性。1，2)连接的葡萄糖残基在天然底物Glc3-Man9-(GlcNAc)2中。
Trimming glucosidase I and II were solubilized from crude calf liver microsomes and partially enriched by a fraction extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific .alpha.-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 .mu.M, 20 .mu.M, 12 .mu.M, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 10 deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I was purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa [kilodalton]. The molecular mass of the native enzyme, determined by gel chromatography, was .apprxeq. 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4.degree. C in the presence of detergent (t1/2 [half-life] .apprxeq. 20 days) and showed high specificity for the hydrolysis of the terminal (.alpha. 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.