Hiraiwa M, Uda Y
Department of Health Chemistry, Niigata College of Pharmacy, Japan.
Jpn J Exp Med. 1988 Jun;58(3):129-38.
Two GM1 ganglioside beta-galactosidases, multimeric form (enzyme I) and monomeric form (enzyme IV), have been purified from bovine liver by the procedures comprising Sephadex G-100 gel filtration, affinity chromatographies on Concanavalin A (Con A)-Sepharose and p-aminophenyl thio-beta-galactoside-CH-Sepharose (PATG-Sepharose) and Sephadex G-200 gel filtration. The multimeric form of the enzyme was purified 13,000-fold and monomeric form was 68,700-fold. On sodium dodecyl sulfate poly acrylamide gel electrophoresis, the monomeric form of the enzyme gave a single protein band with a molecular weight of 65,000, while the multimeric form gave two minor protein bands with molecular weights of 32,000 and 20,000 in addition to the major band at 65,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide. Enzyme I showed a broad pH optimum between pH 4.3 and 5.0, while enzyme IV was most active at pH 4.75. The pI values of beta-galactosidases I and IV were 4.6 and 5.8, respectively. Both enzymes were quite stable upon preincubation at 45 degrees C under acidic condition (pH 4.5), but rapidly lost their activities under neutral condition (pH 7.0). The apparent Km values for GM1 ganglioside of beta-galactosidases I and IV were calculated to be 2.0 x 10(-4) M and 3.3 x 10(-4) M, respectively.
已通过包括Sephadex G - 100凝胶过滤、伴刀豆球蛋白A（Con A）-琼脂糖亲和层析、对氨基苯硫基-β-半乳糖苷-CH-琼脂糖（PATG-琼脂糖）亲和层析以及Sephadex G - 200凝胶过滤的方法，从牛肝中纯化出两种GM1神经节苷脂β-半乳糖苷酶，即多聚体形式（酶I）和单体形式（酶IV）。酶的多聚体形式纯化了13000倍，单体形式纯化了68700倍。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，酶的单体形式呈现一条分子量为65000的单一蛋白条带，而多聚体形式除了在65000处的主要条带外，还给出两条分子量分别为32000和20000的次要蛋白条带。两种酶都能从GM1神经节苷脂和乳糖基神经酰胺上释放出末端半乳糖。酶I在pH 4.3至5.0之间表现出较宽的最适pH值，而酶IV在pH 4.75时活性最高。β-半乳糖苷酶I和IV的pI值分别为4.6和5.8。两种酶在酸性条件（pH 4.5）下于45℃预孵育时相当稳定，但在中性条件（pH 7.0）下会迅速丧失活性。β-半乳糖苷酶I和IV对GM1神经节苷脂的表观Km值经计算分别为2.0×10⁻⁴M和3.3×10⁻⁴M。
