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系统感染TMV的番茄叶片胞外四种#beta#-N-乙酰氨基己糖苷酶的纯化和性质

史益敏, 颜季琼, 费雪南, 许煜泉

上海农学院植物科学系.上海 201101

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#beta#-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(charge isomer), 用过碘酸-Schiff反应证明是糖蛋白,以p-硝基苯-N-乙酰-#beta#-D-氨基葡萄糖苷(pNP-#beta#-D-GlcNAc)和p-硝基苯-N-乙酰-#beta#-D-氨基半乳糖苷(pNP-#beta#-D-GalNAc)为底物,这些酶具有相似的性质。N-乙酰氨基葡萄糖(GlcNAc)和N-乙酰氨基半乳糖(GalNAc)是这些酶的竞争性抑制剂,Ag~(+)和Hg~(2+)是它们的强抑制剂,Fe~(2+)、Fe~(3+)和Cu~(2+)也抑制其活性。

关键词： 番茄; TMV; β－N－乙酰氨基己糖苷酶

Purification and properties of four forms of the apoplastic #beta-N-acetyl-D-hexosaminidase from tomato leaves systemically infected by TMV

Shi Yimin, Yan Jiqiong, Fei Xuenan, Xu Yuquan

Department of Plant Science,Shanghai Agricultural College.Shanghai 201101；China

Abstract

Four forms of the apoplastic #beta#-N-acetyl-D-hexosaminidase were purified from tomato leaves systemically infected with TMV, by -20℃ acetone precipitation, and chromatography through CM-Sephadex C-25 column, DEAE-Sephadex A-25 column and Sephadex G-150 column. The four forms of the enzyme were charge isomers, composed of 75 kD subunits, and glycoproteins. They showed optimal pH between 4.8-5.0 and optimal temerature between 44-47℃. Studies on the thermostability indicated that all four forms of the enzyme had biphasic denaturation curves. With p-nitrophenyl-N-acetyl-#beta#-glucosaminide as substrate, the Kms of forms Ⅰ,Ⅱ,Ⅲ and Ⅳ of the enzyme were 0.32, 0.24, 0.31 and 0.26 mmol/L respectively, and, with p-nitrophenyl-N-acetyl-#beta#-galac-tosaminide as substrate, 0.5, 0.5, 0.5 and 0.34 mmol/L respectively. N-Acetyl-D-glucosamine and N-acetyl D-galactosamine were competitive inhibitors of the enzyme activity. Ag~(+) and Hg~(2+) were potent inhibitors, and Fe~(2+), Fe~(3+) and Cu~(2+) were also inhibitors of the enzyme activity.

Key words: Tomato;Tobacco mosaic virus;#beta#-Nacetyl-D-hexosaminidase

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引用本文：

史益敏, 颜季琼, 费雪南, 许煜泉. 系统感染TMV的番茄叶片胞外四种#beta#-N-乙酰氨基己糖苷酶的纯化和性质[J]. 生理学报 1996; 48 (3): .

Shi Yimin, Yan Jiqiong, Fei Xuenan, Xu Yuquan. Purification and properties of four forms of the apoplastic #beta-N-acetyl-D-hexosaminidase from tomato leaves systemically infected by TMV. Acta Physiol Sin 1996; 48 (3): (in Chinese with English abstract).