Shoup V A, Touster O
J Biol Chem. 1976 Jul 10;251(13):3845-52.
Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.
先前已在大鼠肝脏中鉴定出三种形式的α-D-甘露糖苷酶，每种形式都定位于不同的亚细胞组分：溶酶体、高尔基体膜和胞质溶胶。本通讯报道了胞质溶胶形式的纯化和特性鉴定。借助竞争性抑制剂甘露糖胺和作为稳定剂的二硫赤藓糖醇，该酶以良好的产率纯化了12000倍，纯度约为90%。根据所使用的方法，该酶的分子量在372,000至490,000范围内。由于通过十二烷基硫酸钠聚丙烯酰胺电泳测得亚基分子量为110,000，该酶可能是四聚体。以对硝基苯基-α-D-甘露糖苷为底物时，最适pH值显示为5.5至5.9（在存在1 mM CoCl2的情况下）。观察到正常的米氏动力学，Km为0.14 mM。甘露糖胺是一种竞争性抑制剂，Ki为0.007 mM。在某些条件下由Co2+、Mn2+和Fe2+稳定的纯化酶在低蛋白浓度下不稳定。由于电泳样品显示出阳性过碘酸-希夫染色，该酶可能含有碳水化合物。纯化的胞质溶胶α-D-甘露糖苷酶的可得性现在应该能够进行底物特异性、免疫学和结构研究，这可能有助于阐明该酶的生物学作用。
