Hernandez L M, Ballou L, Ballou C E
Department of Biochemistry, University of California, Berkeley 94720.
Carbohydr Res. 1990 Aug 1;203(1):1-11. doi: 10.1016/0008-6215(90)80040-a.
Oligosaccharides obtained from Saccharomyces cerevisiae mannoproteins by digestion with endo-N-acetyl-beta-D-glucosaminidase H were fractionated by anion-exchange chromatography, by elution with 50-100mM NaOH without or with a sodium-acetate gradient, and detected with a pulsed amperometric detector (PAD). The elution times of homologous oligosaccharides fell on a straight line having a slope characteristic of the structural type. The response of the PAD detector per mole of oligosaccharide increased about 2-fold going from Man3GlcNAc to Man13GlcNAc, and appeared to depend primarily on the oxidation of the reducing-end N-acetylglucosamine unit common to all the oligosaccharides. The digestion of a Man10GlcNAc with jack-bean alpha-mannosidase was monitored by injecting portions of the crude reaction mixture, and the intermediates were characterized by their elution positions and n.m.r. spectra in the anomeric proton region. One commercial jack-bean alpha-mannosidase preparation contained a novel endolytic activity that released N-acetylglucosamine from the reducing ends of the oligosaccharides and was shown to convert P----6 alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc to P----6 alpha Man----6 alpha Man----6 alpha beta Man plus free N-acetylglucosamine. Another commercial jack-bean alpha-mannosidase converted the Man10GlcNAc to a Man3GlcNAc having the structure alpha Man----6 beta Man----4 alpha beta GlcNAc, [formula: see text] whereas the Oerskovia sp. alpha-mannosidase converted the same oligosaccharide to a Man4GlcNAc having the structure alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc. [formula: see text]
用内切-N-乙酰-β-D-氨基葡萄糖苷酶H消化酿酒酵母甘露糖蛋白得到的寡糖，通过阴离子交换色谱法进行分离，用50-100mM NaOH（有无醋酸钠梯度）洗脱，并用脉冲安培检测器（PAD）检测。同源寡糖的洗脱时间落在具有结构类型特征斜率的直线上。从Man3GlcNAc到Man13GlcNAc，每摩尔寡糖的PAD检测器响应增加约2倍，并且似乎主要取决于所有寡糖共有的还原端N-乙酰葡糖胺单元的氧化。通过注入部分粗反应混合物监测用刀豆α-甘露糖苷酶消化Man10GlcNAc的过程，中间体通过其洗脱位置和端基质子区域的核磁共振光谱进行表征。一种市售的刀豆α-甘露糖苷酶制剂具有一种新的内切活性，可从寡糖的还原端释放N-乙酰葡糖胺，并显示将P----6αMan----6αMan----6βMan----4αβGlcNAc转化为P----6αMan----6αMan----6αβMan加游离N-乙酰葡糖胺。另一种市售的刀豆α-甘露糖苷酶将Man10GlcNAc转化为具有αMan----6βMan----4αβGlcNAc结构的Man3GlcNAc，[化学式：见正文]，而Oerskovia sp.α-甘露糖苷酶将相同的寡糖转化为具有αMan----6αMan----6βMan----4αβGlcNAc结构的Man4GlcNAc。[化学式：见正文]
