Szumilo T, Kaushal G P, Hori H, Elbein A D
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284.
Plant Physiol. 1986 Jun;81(2):383-9. doi: 10.1104/pp.81.2.383.
The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [(3)H]mannose from the [(3)H]Man(9)GlcNAc as well as for releasing mannose from p-nitrophenyl-alpha-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca(2+) was the most effective cation for reversing this inhibition. Mn(2+) was considerably less effective than Ca(2+) and Mg(2+) was without effect. The processing mannosidase was inhibited by alpha1,2- and alpha1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and alpha1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [(3)H]Man(9)GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man(5)GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific alpha1,2-mannosidase.
绿豆幼苗的微粒体部分含有甘露糖苷酶活性，能够从[(3)H]Man(9)GlcNAc中水解[(3)H]甘露糖，也能从对硝基苯基-α-D-甘露吡喃糖苷中释放甘露糖。糖蛋白加工甘露糖苷酶用1.5% Triton X-100从微粒体中溶解出来，并通过常规方法以及在甘露聚糖-琼脂糖和甘露糖胺-琼脂糖上的亲和层析纯化了130倍。最终的酶制剂含有微量的芳基甘露糖苷酶，但这种活性被苦马豆素抑制，而加工酶不受影响。加工酶的最适pH为5.5至6.0，在0.1% Triton X-100存在下活性最佳。该酶被乙二胺四乙酸抑制，而Ca(2+)是逆转这种抑制作用最有效的阳离子。Mn(2+)的效果比Ca(2+)差得多，Mg(2+)则没有作用。加工甘露糖苷酶被α1,2-和α1,3-连接的甘露糖寡糖抑制（3毫摩尔时50%抑制），而游离甘露糖和α1,6-连接的甘露糖寡糖则无效。甘露糖胺也是这种酶的抑制剂。芳基甘露糖苷酶和加工甘露糖苷酶也可以通过它们对各种加工抑制剂的敏感性来区分。芳基甘露糖苷酶被苦马豆素和1,4-二脱氧-1,4-亚氨基-D-甘露糖醇抑制，但不被脱氧野尻霉素或其他抑制剂抑制，而加工甘露糖苷酶仅被脱氧野尻霉素抑制。加工甘露糖苷酶与[(3)H]Man(9)GlcNAc长时间孵育，产物通过凝胶过滤鉴定。即使孵育24小时后，仅有的两种放射性产物是Man(5)GlcNAc和游离甘露糖。因此，这种酶似乎与动物加工酶甘露糖苷酶I相似，显然是一种特异性的α1,2-甘露糖苷酶。
