Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of beta-glucosidase.
Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of beta-glucosidase.
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DOI：
10.1016/0003-9861(74)90262-8
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发表时间：
1973-09
期刊：
影响因子：
--
通讯作者：
M. Rudick;A. Elbein
中科院分区：
文献类型：
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作者：
M. Rudick;A. Elbein
采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和分析超速离心法对烟曲霉胞外α-葡萄糖苷酶(Mr 63,560)进行了纯化。以麦芽糖为底物，该酶的最适pH为4.5，KmandV值为1.85mM，最适μ摩尔浓度为10min/mg。它是α构型所特有的，很容易被麦芽糖和异麦芽糖水解，但利用海藻糖和对-硝基-α-d-葡萄糖苷的速度很慢。1摩尔的酶含有55.1摩尔的甘露糖、3.2摩尔的氨基葡萄糖和5.4摩尔的葡萄糖。然而，三氯乙酸沉淀释放了所有的葡萄糖，而甘露糖和氨基葡萄糖仍然与蛋白质结合，表明后者是共价结合到酶上的。在[~3H]NaBH_4存在下用0.05nNaOH处理该酶，不释放低聚糖，也不产生氚已糖醇。然而，在[~3H]NaBH_4存在下，较强的碱性处理(1nNaOH，100℃，6小时)释放出低聚糖，导致[~3H]氨基葡萄糖醇的形成。这些数据表明了氨基葡萄糖→天冬酰胺的连接。α-葡萄糖苷酶与β-N-乙酰氨基葡萄糖苷酶作用后，可释放低聚糖链(S)，提示为一个Man(GlcNAc)2Asn连锁。
An extracellular α-glucosidase (Mr63,560) fromAspergillus fumigatuswas purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and analytical ultracentrifugation. The enzyme had a pH optimum of 4.5,KmandVvalues of 1.85 mmand 10 μmoles/min/mg with maltose as substrate. It was specific for the α-configuration, readily hydrolyzed maltose and isomaltose, but utilized trehalose andp-nitrophenyl-α-d-glucoside very slowly. One mole of enzyme contained 55.1 moles of mannose, 3.2 moles of glucosamine, and 5.4 moles of glucose. However, precipitation with trichloroacetic acid released all of the glucose, while mannose and glucosamine remained associated with the protein, indicating that the latter sugais were covalently bound to the enzyme. When the enzyme was treated with 0.05nNaOH in the presence of [3H]NaBH4, no oligosaccharide was released and no tritiated hexitols were observed. However, stronger alkaline treatment (1nNaOH, 100 °C, 6 hr) in the presence of [3H]NaBH4released the oligosaccharide and led to formation of [3H]glucosaminitol. These data suggested a glucosaminyl → asparagine linkage. Treatment of the α-glucosidase with endo-β-N-acetylglucosaminidase caused the release of oligosaccharide chain(s), indicating a Man(GlcNAc)2Asn linkage.