摘要
系统感染ＴＭＶ的番茄叶胞外提取液经冰冻干燥浓缩、－２０℃丙酮沉淀、离子交换柱层析和凝胶柱层析纯化，获得４种β－Ｎ－乙酰氨基己糖苷酶。这些酶是由７５ｋＤ亚基构成的电荷异构体（ｃｈａｒｇｅｉｓｏｍｅｒ），用过碘酸－Ｓｃｈｉｆｆ反应证明是糖蛋白，以Ｄ－硝基苯－Ｎ－乙酰－β－Ｄ－氨基葡萄糖苷（ｐＮＰ－β－Ｄ－ＧｌｃＮＡｃ）和ｐ－硝基苯－Ｎ－乙酰－β－Ｄ－氨基半乳糖苷（ｐＮＰ－β－Ｄ－ＧａｌＮＡｃ）为底物，这些酶具有相似的性质。Ｎ－乙酸氨基葡萄糖（ＧｌｃＮＡｃ）和Ｎ－乙酰氨基半乳糖（ＧａｌＮＡｃ）是这些酶的竞争性抑制剂，Ａｇ＋和Ｈｇ２＋是它们的强抑制剂，Ｆｅ２＋、Ｆｅ３＋和Ｃｕ２＋也抑制其活性。
Four forms of the apoplastic β-Nacetyl-D-hexosaminidase were purifiedfrom tomato leaves systemically infected with TMV, by-20℃ acetone precipitation, and chromatography throughCM-Sephadex C-25 column, DEAESephadex A-25 column and SephadexG-150 column. The four forms of theenzyme were charge isomers, composedof 75 kD subunitS, and glycoproteins.They showed optimal PH between 4. 8-5. 0 and optimal temperature between44 - 47℃. Studies on the thermostubility indicated that all four forms ofthe enzyme had biphasic denaturationcurves. With p-nitrophenyl-N- acetylsβ-glucosaminide as substrate, the Kmsof forms Ⅰ, Ⅱ, Ⅲ and Ⅳ of the enzyme were 0. 32, 0. 24, 0. 31 and0. 26 mmol/L respectively, and, withp-nitrophenyl-N-acetyl-β-gatosaminide as substrate, 0. 5, 0. 5, 0. 5and 0. 34 mmol/L respectively. NAcetyl-D-glucoSamine and N-acetyl Dgalactosamine were competitive inhibitors of the enzyme activity. Ag+ andHg2+ were potent inhibitors, and Fe2+,Fe3+and CuZ+were also inhibitors of theenzyme activity.
