Altmann F, März L
Institut für Chemie der Universität für Bodenkultur Wien, Vienna.
Glycoconj J. 1995 Apr;12(2):150-5. doi: 10.1007/BF00731359.
The occurrence of alpha-D-mannosidase II activity in insect cells was studied using pyridylaminated oligosaccharides as substrates and two-dimensional HPLC and glycosidase digestion for the analysis of products. GlcNAcMan5GlcNAc2 was converted to GlcNAcMan3GlcNAc2 by each of the three cell lines investigated (Bm-N, Sf-21, and Mb-0503). The respective activity was highest in Bm-N cells which were used for further experiments. Man5GlcNAc2 was not degraded by the Bm-N cell homogenate. Thus, this alpha-mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi alpha-mannosidase II. The alpha-mannosidase II-like activity from Bm-N cells exhibits a pH optimum of 6.0-6.5, has no requirement for divalent metal ions, and is highly sensitive to swainsonine. The alpha 1,6-linked mannosyl residue is removed first as deduced from the elution time on reversed phase HPLC of the intermediate product. The same branch preference was found with alpha-mannosidase II from mung bean seedlings and Xenopus liver. Upon ultracentrifugation of Bm-N cell homogenate, 72% of the mannosidase acting on the GlcNAcMan5GlcNAc2 substrate was found in the microsomal pellet indicating the enzyme to be membrane-bound.
以吡啶基化寡糖为底物，采用二维高效液相色谱和糖苷酶消化法分析产物，研究了昆虫细胞中α-D-甘露糖苷酶II活性的发生情况。在所研究的三种细胞系（Bm-N、Sf-21和Mb-0503）中，GlcNAcMan5GlcNAc2均被转化为GlcNAcMan3GlcNAc2。在用于进一步实验的Bm-N细胞中，相应活性最高。Bm-N细胞匀浆不能降解Man5GlcNAc2。因此，这种α-甘露糖苷酶与哺乳动物和植物高尔基体α-甘露糖苷酶II基本具有相同的底物特异性。来自Bm-N细胞的α-甘露糖苷酶II样活性的最适pH为6.0-6.5，不需要二价金属离子，并且对苦马豆素高度敏感。从中间产物在反相高效液相色谱上的洗脱时间推断，α-1,6-连接的甘露糖残基首先被去除。绿豆幼苗和非洲爪蟾肝脏中的α-甘露糖苷酶II也有相同的分支偏好。对Bm-N细胞匀浆进行超速离心后，发现作用于GlcNAcMan5GlcNAc2底物的甘露糖苷酶72%存在于微粒体沉淀中，表明该酶是膜结合的。
