Purification and Characterization of a Lectin from Dictyophora indusiata Mycelia
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摘要:
目的 凝集素是竹荪的一类生理活性物质，为进一步明确其药用功效，开展了长裙竹荪凝集素理化性质研究。 方法 长裙竹荪菌丝体通过生理盐水抽提、20%～75%饱和度的硫酸铵沉淀、DEAE-Sepharose和SephadexG-100柱层析纯化得到长裙竹荪菌丝体凝集素（DIL）；并通过血细胞凝集活性、糖抑制试验、酸碱稳定性、热稳定性分析以及金属离子等因素来测定长裙竹荪菌丝体凝集素的理化性质。 结果 经PAGE显示单一条带，测得其亚基相对分子质量为43.6 kDa。该凝集素对供试的鸽、兔、鸭和鸡血红细胞具有凝集作用，对兔红细胞的凝集作用可被D-半乳糖和N-乙酰胺基-葡萄糖所抑制。长裙竹荪凝集素对热不稳定，经50℃处理10 min，活性明显降低；该凝集素在pH 4.0～8.0都保持着较高的凝集活性，其凝血活性依赖于Ca2+和Mg2+二价金属离子，Mn2+和Zn2+则无影响。 结论 长裙竹荪菌丝体凝集素对动物血红细胞具有较好的凝集活性，而且凝集活性需要在一定浓度的Ca2+和Mg2+才会起作用。 Abstract:Objective Physicochemical properties of the physiologically active lectin component in Dictyophora indusiata Fisscher, a popular edible mushroom in China, were studied to determine the medicinal function. Method A lectin compound was isolated and purified from the mushroom mycelia using physiological saline extraction followed by precipitation in a 20%~75 % (NH4)2SO4 solution and purification with DEAE-Sephrose and Sephadex G-100 gel filtration chromatography. Physicochemical properties of the purified mycelial lectin, DIL, were characterized through the analyses of hemagglutination activity, sugar inhibition, pH and thermal stabilities, and interaction with metal ions. Results DIL showed one band in SDS-PAGE and a molecular weight of 43.6 kDa. It could agglutinate the erythrocytes from pigeon, rabbit, duck, and chicken. The hemagglutination on pigeon erythrocytes was inhibited by D-galactose and N-acetyl-amino-glucose. Being heat-unstable, DIL declined in agglutinating activity significantly at 50℃ in 10min. It remained highly active within pH 4.0~8.0 and was affected by Ca2+ or Mg2+ but not Mn2+ or Zn2+. Conclusion It appeared that DIL was hemagglutination-active in the presence of Ca2+ and/or Mg2+ on animal erythrocytes. -
Key words:
- Lectin /
- Dictyophora indusiata Fisscher /
- isolation and purification /
- characterization
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图 1 长裙竹荪菌丝体凝集素的DEAE-Sepharose Fast Flow层析
Figure 1. DEAE-Sepharose fast flow chromatography of DIL
表 1 长裙竹荪菌丝体凝集素的凝血活性
Table 1. Agglutinating activity of DIL
血细胞 Hemocyte 凝集活性 Agglutinating activity 鸽血 Pigeon blood 210 兔血 Rabbit blood 29 鸭血 Duck blood 28 鸡血 Chicken blood 23 
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表 2 长裙竹荪菌丝体凝集素对单糖的抑制作用
Table 2. Inhibition of DIL agglutinating activity by sugars
单糖种类 Monosaccharide types 凝集活性 Agglutinating activity D-半乳糖 D-galactose 23 D-甘露糖 D-mannose 210 D-海藻糖 D-trehalose 210 L-鼠李糖 L-rhamnose 210 L-阿拉伯糖 L-arabinose 210 N-乙酰胺基-葡萄糖 N-acetamino-glucose 25 注：样品起始质量浓度为10 mg·mL−1。
Note: The initial concentration of the sample was 10 mg·mL−1.
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表 3 pH对长裙竹荪菌丝体凝集素凝集活性的影响
Table 3. Effect of pH on DIL agglutinating activity
pH 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 凝集活性 Agglutinating activity 28 211 211 210 210 210 28 27
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表 4 温度对长裙竹荪菌丝凝集活性的影响
Table 4. Effect of temperature on DIL agglutinating activity
温度Temperature 长裙竹荪菌丝体凝集素含量
Lectin content of Dictyophora indusiata mycelium/（μg·mL−1）1 000 500 250 125 62.5 30℃ + + + + + 40℃ + + + + + 50℃ + + + − − 60℃ + + + − − 70℃ − − − − − 80℃ − − − − − 90℃ − − − − − 100℃ − − − − − 注：“+” 凝集；“−” 不凝集。
Note: “+” indicates agglutination, “−”no agglutination.
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表 5 金属离子对长裙竹荪菌丝体凝集素凝集活性的影响
Table 5. Effect of metal ions on DIL agglutinating activity
处理方式 Treatment 凝集活性 Agglutinating activity 未经处理 Untreated 29 经EDTA处理 EDTA treated − 处理后加10 mmol·L−1 Ca2+
After treatment with 10 mmol·L−1 Ca2+28 处理后加10 mmol·L−1L Zn2+
After treatment with 10 mmol·L−1 Zn2− 处理后加10 mmol·L−1 Mg2+
After treatment with 10 mmol·L−1 Mg2+27 处理后加10 mmol·L−1 Mn2+
After treatment with 10 mmol·L−1 Mn2+− 注：凝集素初始质量浓度10 mg·mL−1；“−” 不凝集。
Note: Initial DIL concentration=10 mg·mL−1; “−” indicates no agglutination.
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