Studies on Human β-d-N-Acetylhexosaminidases I. PURIFICATION AND PROPERTIES
Studies on Human β-d-N-Acetylhexosaminidases I. PURIFICATION AND PROPERTIES
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人 β-d-N-乙酰己糖胺酶的研究 I. 纯化和性质
DOI：
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发表时间：
1974
期刊：
影响因子：
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通讯作者：
E. Beutler
中科院分区：
文献类型：
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作者：
S. Srivastava;Y. Awasthi;A. Yoshida;E. Beutler
从人胎盘中分离纯化了β-d-N-乙酰氨基己糖苷酶A和B。在纯化的初始步骤中，即提取、硫酸铵沉淀、冻干、Sephadex G-200过滤和DEAE-纤维素柱层析，氨基己糖苷酶A和氨基己糖苷酶B被一起纯化。通过第二次DEAE-纤维素柱层析，然后是ECTEOLA-纤维素柱层析，Sephadex G-200过滤，电聚焦和第三次Sephadex G-200过滤进一步纯化己糖胺酶A。通过CM-纤维素柱层析、电聚焦和Sephadex-G-200过滤进一步纯化氨基己糖苷酶B。在第一次Sephadex G-200过滤后，还通过第二种方法纯化氨基己糖苷酶A和B。该方法包括磷酸钙凝胶、DEAE-Sephadex、ECTEOLA-纤维素、CM-Sephadex和CM-纤维素柱层析和制备聚丙烯酰胺圆盘电泳。纯化的氨基己糖苷酶A和氨基己糖苷酶B在聚丙烯酰胺圆盘电泳上移动为具有几乎所有酶活性的单一蛋白条带。氨基己糖苷酶A和B在含有0.1 m（NH 4）2SO 4的10 mm磷酸盐缓冲液中在4 ℃下至少稳定4个月。发现氨基己糖苷酶A和氨基己糖苷酶B的等电点分别为pH 5.4和7.9。胎盘氨基己糖苷酶被发现与肝脏和成纤维细胞酶在免疫学和抗原性上不同。与其他人的报道相反，神经氨酸酶不能将热不稳定的氨基己糖苷酶A转化为类似于氨基己糖苷酶B的热稳定形式的酶。
Abstract β-d-N-Acetylhexosaminidase A and B were purified from human placenta. In the initial steps of purification, namely, extraction, ammonium sulfate precipitation, lyophilization, Sephadex G-200 filtration, and DEAE-cellulose column chromatography hexosaminidase A and hexosaminidase B were purified together. Hexosaminidase A was further purified by a second DEAE-cellulose column chromatography followed by ECTEOLA-cellulose column chromatography, Sephadex G-200 filtration, electrofocusing, and a third Sephadex G-200 filtration. Hexosaminidase B was further purified by CM-cellulose column chromatography, electrofocusing, and Sephadex-G-200 filtration. After the first Sephadex G-200 filtration hexosaminidase A and B were also purified by a second method. This method involved calcium phosphate gel, DEAE-Sephadex, ECTEOLA-cellulose, CM-Sephadex and CM-cellulose column chromatography, and preparative polyacrylamide disc electrophoresis. Purified hexosaminidase A and hexosaminidase B moved on polyacrylamide disc electrophoresis as single protein bands with virtually all of the enzyme activity. Hexosaminidase A and B in 10 mm phosphate buffer containing 0.1 m (NH4)2SO4 were stable at 4° for at least 4 months. The isoelectric points of hexosaminidase A and hexosaminidase B were found to be pH 5.4 and 7.9, respectively. Placental hexosaminidases were found to differ electrophoretically and antigenically from the liver and fibroblast enzymes. In contrast to the reports of others neuraminidase failed to convert heat-labile hexosaminidase A to a heatstable form of enzyme resembling hexosaminidase B.