Yamashiro K, Itoh H, Yamagishi M, Natsuka S, Mega T, Hase S
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka.
J Biochem. 1997 Dec;122(6):1174-81. doi: 10.1093/oxfordjournals.jbchem.a021878.
Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.
中性α-甘露糖苷酶从母鸡输卵管中纯化至同质。该酶在凝胶过滤中的分子量为480 kDa，在2-巯基乙醇存在下的SDS-PAGE上的110-kDa条带表明它由四个亚基组成。活化的酶可水解对硝基苯基α-D-甘露糖苷和高甘露糖型糖链。这种底物特异性与日本鹌鹑输卵管中的中性α-甘露糖苷酶报道的几乎相同[Oku和Hase（1991年）《生物化学杂志》110，982-989]。Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc（Km =0.44 mM）的水解速度比Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcbeta1-4GlcNAc快四倍，并且在与活化的α-甘露糖苷酶消化时，Manα1-6(Manα1-2Manα1-2Manα1-3)++ +Manβ1-4GlcNAc作为来自Man9GlcNAc的终产物获得。该酶在与Co2+预孵育时被激活24倍。用其他金属离子如Mn2+、Ca2+、Fe2+、Fe3+和Sr2+激活的程度小于5倍，而Zn2+、Cu2+和Hg2+抑制酶活性。酶活性和用Co2+激活的最佳pH值均约为7。纯化的、经EDTA处理的和经Co2+激活的酶的钴离子含量分别为每分子1.5、0.0和3.9。由于经Co2+激活的酶在与EDTA孵育时逐渐失去活性，并且在添加Co2+后活性迅速恢复，因此Co2+与酶的结合似乎对其激活至关重要。蛋白酶抑制剂的结果以及激活前后SDS-PAGE的结果表明，报道的猴脑α-甘露糖苷酶激活中的蛋白水解切割似乎不涉及。
