McCabe N R, Dawson G
Department of Pediatrics, University of Chicago, IL 60637.
Biochim Biophys Acta. 1991 Apr 8;1077(2):133-40. doi: 10.1016/0167-4838(91)90049-6.
Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.
溶酶体β-甘露糖苷酶（EC 3.2.11.25）的功能大小为120 - 150 kDa，但据报道，从豚鼠肝脏（GPL）中纯化得到的该酶呈现出一条对应分子量为110 kDa的单一条带。为了研究β-甘露糖苷酶的亚基结构和组织特异性表达，我们在兔体内制备了针对GPL β-甘露糖苷酶的多克隆抗体，该抗体能免疫沉淀β-甘露糖苷酶活性，且不含有其他溶酶体水解酶活性。在 -20℃保存并在2-巯基乙醇存在下进行SDS聚丙烯酰胺凝胶电泳后，纯化的GPL β-甘露糖苷酶样品在97 kDa处出现一条主要的考马斯亮蓝染色带。蛋白质免疫印迹分析证实了这一点，该分析还显示出一条迁移速度更快的37 kDa蛋白质条带。相比之下，在蛋白酶抑制剂存在下制备的新鲜GPL匀浆的蛋白质免疫印迹分析显示，主要条带位于150 kDa处。经过冻融处理后，我们观察到120 kDa和20 kDa的免疫反应条带，最终观察到97 kDa、37 kDa和20 kDa的免疫反应条带。通过正丁醇/乙醚提取相关脂质，可加速150 kDa形式形成97 kDa、37 kDa和20 kDa形式，这表明这些亚基之间存在紧密的疏水相互作用。与肝脏不同，豚鼠肾脏（GPK）的新鲜和冻融制剂均仅产生97 kDa、37 kDa和20 kDa的亚基形式，这证实了这些是主要的β-甘露糖苷酶亚基。内切糖苷酶F处理可将肝脏和肾脏中的97 kDa形式转化为91 kDa形式，将37 kDa形式转化为35 kDa形式，而20 kDa形式不受影响。用合成底物4MU-β-甘露糖苷测定的总β-甘露糖苷酶活性不受150 kDa形式解离为97 kDa、37 kDa和20 kDa亚基的影响，这表明这些是该酶的功能形式，而非蛋白水解降解产物。
