Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.
Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.
复制标题
人酸性 β-葡萄糖苷酶：在 N-烷基-脱氧野尻霉素-琼脂糖上亲和纯化正常胎盘和戈谢病脾酶。
DOI：
10.1159/000469336
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Grabowski,GA
中科院分区：
文献类型：
--
作者：
Osiecki-Newman,KM;Fabbro,D;Dinur,T;Boas,S;Gatt,S;Legler,G;Desnick,RJ;Grabowski,GA
用琼脂糖结合的1-脱氧诺吉霉素N-烷基衍生物N-(9-羧基)-和N-(L L-羧基十一基)-脱氧诺吉霉素亲和纯化了正常来源和1型德系犹太人高谢病来源的酸性β-葡萄糖苷酶。这些不可降解的缓蚀剂载体的容量分别为0.5mgNormal-GLC/ml和0.75mgg/ml沉淀胶。纯化的正常酶(产率14-18%)比活力为1.6×10~(-6)nmol/h/mg蛋白质，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和反相高效液相色谱柱层析，均一种M(R)=67000的蛋白质。测序结果显示该基因只有一个N末端，其前22个N末端氨基酸的序列与该基因预测的序列具有共线关系。氨基酸组成分析表明，其疏水性氨基酸含量较高(35%)。N-十烷基脱氧诺吉霉素载体促进了从1型AJGD脾中提取的残留酶的纯化，分四步进行了约7,500倍的纯化，产率约为11%。这些新的亲和载体提供了比其他亲和或高效液相色谱方法更好的稳定性、容量和/或特异性来纯化这种溶酶体糖苷酶。
Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)-and N-(l l-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid ß-glucosidase (ß-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease(AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal ß-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10^6 nmol/h/mg protein and was homogeneous as evidenced by a single protein species of M(r) = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography(HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the ß-Glc cDNA. Amino acid composition analyses of ß-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11 %. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.