牛舌菌凝集素的分离、性质及细胞凝集活性

ISOLATION,CHARACTERIZATION AND HAEMAGGLUTINATION OF THE LECTIN FROM FISTULINA HEPATICA

牛舌菌子实体经生理盐水抽提、硫酸铵沉淀、DEAE Cellulose和SephadexG 10 0柱层析纯化得到牛舌菌凝集素 (FistulinahepaticaLectin ,FHL)。FHL经PAGE显示单一条带 ,SDSPAGE测得其亚基相对分子质量为 6 3.6KD ,SephadexG 10 0凝胶过滤测得相对分子质量为 6 3KD ,FHL中性糖含量为 9.7% ,IEF PAGE测得其等电点为 5 .36。该凝集素对人的A、B、AB和O型血细胞具有凝集作用 ,对兔红细胞的凝集作用可被乳糖和N 乙酰半乳糖胺所抑制。氨基酸组成分析表明 ,FHL含有 16种氨基酸 ,其中天冬氨酸和谷氨酸含量较高。FHL对热、酸具有一定的稳定性 ,经 6 0℃处理 10min ,仍有较高的活性 ,在 pH 3.0～ 6 .0范围内较稳定 ,但在碱性pH环境中 (pH 7.0～ 10 .8) ,FHL的凝集活性下降明显。β 消去反应测得其糖和蛋白质的连接键为O 型糖肽键。抗肿瘤活性测定表明 ,FHL对HeLa细胞具有明显抑制作用。

The lectin FHL was isolated and purified from the fruiting bodies of Fistulina hepatica by physiological saline extraction,precipitation of 60%(NH 4) 2SO 4 and DEAE-Cellulose,Sephadex G-100 chromatography.It showed single band in PAGE identification.The relative molecular mass of FHL was 63 KD tested by gel filtration,the subunit of FHL was 63.6 KD determined by SDS-PAGE.FHL contained 9.7% neutral saccharide,its pI was about 5.36 by isoelectric focusing.This lectin agglutinated rabbit erythrocytes and human erythrocytes,the hemagglutination of the lectin on rabbit erythrocytes was inhibited by lactose and GalNAc.Amino acid composition analysis showed that it contained 16 kinds of amino acid,in which the content of aspartic acid and glutamic acid was relatively high.FHL was certain stable in acid and heat treatment.It still kept partial activity after being heated for 10 min at 60 ℃,and had high activity within pH 3.0～6.0,but the agglutinating activity of FHL declined obviously at the range of pH 7.0～10.8.β-Elimination revealed the bond between the polysaccharide and the protein of FHL was O-type glucopeptide linkage.FHL had inhibiting effect on HeLa cells in vitro.