短裙竹荪(Dityophora duplicata)凝集素纯化与生化性质

Purification and Characterization of Lectin from Dityophora duplicata

短裙竹荪子实体经生理盐水抽提、硫酸铵沉淀、DEAE Sepharose和SephadexG 10 0柱层析纯化得到短裙竹荪凝集素 (Dityophoraduplicata(Bosc)Fischerlectin) ,简称DDFL .DDFL经PAGE显示单一条带 ,SDS PAGE测得其亚基分子量为 2 2 3kD ,SephadexG 10 0凝胶过滤测得分子量为 4 5 3kD ,DDFL不含中性糖 ,IEF测得其等电点为 3 92 .该凝集素对供试的 4种血型人血和兔、小牛、鸭、鸡、鲫鱼以及青蛙血红细胞具有凝集作用 ,但不凝集鳖红细胞 .它还可以凝集小鼠脾脏淋巴细胞和小鼠S180 肉瘤细胞 ,对兔红细胞的凝集作用可被乳糖、棉子糖、半乳糖、α 甲基半乳糖、β 甲基半乳糖和N 乙酰半乳糖胺所抑制 .氨基酸组成分析表明 ,DDFL含有 17种氨基酸 ,其中天冬氨酸、丝氨酸、苯丙氨酸和丙氨酸含量较高 .经测定 ,其N末端为甘氨酸 .DDFL对热、酸和碱具有一定的稳定性 ,经 6 0℃处理 10min ,可保持较高的活性 ,在pH 4 0～ 9 0范围内较稳定 ,其凝血活性依赖于Mg2 + 和Ca2 + 二价阳离子 ,Mn2 + 和Zn2 + 则无影响 .DDFL对小鼠腹腔注射的半致死量为 70 6 3mg kg .

The lectin DDFL from the fruiting bodies of Dityophora duplicata(Bosc) Fischer was isolated and purified by physiological saline extraction, (NH 4) 2SO 4 precipitation and DEAE Sepharose, Sephadex G 100 chromatography purification. It turned out to be a single band on PAGE. The relative molecular mass of DDFL was 45.3 kD tested by gel filtration,and the subunit was 22 3 kD determined by SDS PAGE, its pI was about 3 92 by isoelectrio focusing. This lectin could agglutinate erythrocytes from rabbit, calf, duck, chicken, crucian, frog and 4 types of human erythrocytes, but did not agglutinate erythrocytes from turtle. It also agglutinated the peripheral lymphocytes cells and S 180 tumor cells from mouse. The hemagglutination on rabbit erythrocytes was inhibited by lactose, raffinose,galactose,α methyl galactose,β methyl galactose and N acetyl galactosamine. The amino acid composition analysis showed that it contained 17 kinds of amino acid, in which the contents of aspartic acid, serine, phenylalanine and alanine were relatively high. The N terminal of DDFL was glycine. DDFL had certain thermal stable, acid stable and alkali stable, it still had high activity after being heated 10 minutes in 60℃, and had high activity within pH 4 0～9 0. Its activity was depended on Mg 2+ and Ca 2+ , not on Mn 2+ and Zn 2+ . DDFL was toxic to mouse by celiac injection with a LD 50 of 70 63 mg/kg.