Tulsiani D R, Touster O
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
J Biol Chem. 1988 Apr 15;263(11):5408-17.
Rat liver Golgi membranes contain two alpha 1,2-specific mannosidases (IA and IB) (Tulsiani, D. R. P., Hubbard, S. C., Robbins, P. W., and Touster, O. (1982) J. Biol. Chem. 257, 3660-3668). Mannosidase IA has now been purified to apparent homogeneity by detergent extraction and (NH4)2SO4 precipitation, followed by Sephacryl S-300, ion-exchange, and hydroxylapatite chromatography. The enzyme was homogeneous by nondenaturing polyacrylamide gel electrophoresis with different gel concentrations, and Ferguson plot analysis indicated an Mr of 230,000 for the native enzyme. Although electrophoresis under denaturing conditions generally gave a subunit Mr of 57,000, electrophoresis of less than 1 microgram of protein yielded a faint doublet of Mr 57,000 and 58,000. Thus, the enzyme appears to be a tetramer with four very similar subunits. The enzyme bound to concanavalin A-Sepharose 4B only when it was kept in contact with the lectin for 16 h. Endoglycosidase H treatment resulted in loss of its binding to the lectin, without leading to a detectable change in the size of the enzyme subunit. On electrophoretic gels, the enzyme gave a faint positive stain with periodic acid-Schiff's base. The enzyme contained about 0.9% hexose by direct analysis. It did not bind to affinity resins specific for neuraminic acid, galactose, or N-acetylglucosamine. All these studies suggest that the enzyme is a glycoprotein containing only one or two clusters of high mannose oligosaccharide. Mannosidase IA is active toward oligosaccharides containing alpha 1,2-linked mannosyl residues. [3H]Man9GlcNAc, [3H] Man8GlcNAc, [3H]Man7GlcNAc, and [3H]Man6GlcNAc are good substrates. Man9GlcNAc, the best substrate, yields Man8, Man7, and Man6 derivatives with structures suggesting that the sequence of release of mannose residues is rather specific. Immunoprecipitation studies using polyclonal antibody (IgG) prepared against homogeneous mannosidase IA cross-reacted with mannosidase IB, a result suggesting that these two enzymes share antigenic determinants. However, no cross-reactivity was observed with rat liver cytosolic and lysosomal alpha-D-mannosidases or with Golgi mannosidase II.
大鼠肝脏高尔基体膜含有两种α1,2-特异性甘露糖苷酶（IA和IB）（图尔西亚尼，D.R.P.，哈伯德，S.C.，罗宾斯，P.W.，和图斯特，O.（1982年）《生物化学杂志》257卷，3660 - 3668页）。甘露糖苷酶IA现已通过去污剂提取和硫酸铵沉淀，随后经Sephacryl S - 300、离子交换和羟基磷灰石柱色谱法纯化至表观均一。该酶在不同凝胶浓度的非变性聚丙烯酰胺凝胶电泳中表现为均一，弗格森图分析表明天然酶的Mr为230,000。虽然在变性条件下电泳通常得到亚基Mr为57,000，但小于1微克蛋白质的电泳产生了微弱的Mr为57,000和58,000的双峰。因此，该酶似乎是由四个非常相似的亚基组成的四聚体。该酶只有在与伴刀豆球蛋白A - Sepharose 4B接触16小时后才会与之结合。内切糖苷酶H处理导致其与凝集素的结合丧失，但未导致酶亚基大小出现可检测到的变化。在电泳凝胶上，该酶用高碘酸 - 希夫碱染色呈微弱阳性。通过直接分析，该酶含有约0.9%的己糖。它不与针对神经氨酸、半乳糖或N - 乙酰葡糖胺的亲和树脂结合。所有这些研究表明该酶是一种糖蛋白，仅含有一或两个高甘露糖寡糖簇。甘露糖苷酶IA对含有α1,2 - 连接甘露糖残基的寡糖有活性。[3H]Man9GlcNAc、[3H]Man8GlcNAc、[3H]Man7GlcNAc和[3H]Man6GlcNAc是良好的底物。最佳底物Man9GlcNAc产生Man8、Man7和Man6衍生物，其结构表明甘露糖残基的释放顺序相当特异。使用针对均一甘露糖苷酶IA制备的多克隆抗体（IgG）进行的免疫沉淀研究与甘露糖苷酶IB发生交叉反应，这一结果表明这两种酶共享抗原决定簇。然而，未观察到与大鼠肝脏胞质和溶酶体α - D - 甘露糖苷酶或高尔基体甘露糖苷酶II的交叉反应。
