Sopher B L, Traviss C E, Cavanagh K T, Jones M Z, Friderici K H
Department of Pathology, Michigan State University, East Lansing 48824.
Biochem J. 1993 Jan 15;289 ( Pt 2)(Pt 2):343-7. doi: 10.1042/bj2890343.
Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.
通过包括伴刀豆球蛋白A-琼脂糖、免疫亲和、TSK-丁基和高效液相色谱阳离子交换色谱在内的四步纯化程序，从牛肾中以24%的产率将溶酶体β-甘露糖苷酶纯化了160,000倍。当通过SDS/PAGE分析并用考马斯亮蓝或银染检测时，纯化的酶制剂由两条主要肽段（100 kDa和110 kDa）和第三条次要肽段（84 kDa）组成。这三条肽段在免疫学上相关，并且在所有色谱步骤中都始终与β-甘露糖苷酶活性相关。从84、100和110 kDa肽段上去除N-连接的碳水化合物后，它们的分子大小分别降至75、86和91 kDa。从患有β-甘露糖苷贮积症的犊牛获得的缺乏β-甘露糖苷酶活性的牛肾，通过单克隆和多克隆抗体反应性判断，不含有可检测量的三种β-甘露糖苷酶肽段。
