中华鳖肝金属硫蛋白的分离、纯化及鉴定(英文)

ISOLATION, PURIFICATION AND IDENTIFICATION OF METALLOTHIONEIN IN CHINESE SOFT-SHELLED TURTLE (PELODISCUS SINENSIS) LIVER

金属硫蛋白 (metallothionein ,MT)是一类低分子量、富含半胱氨酸的金属结合蛋白。MT几乎广泛分布于所有生物 ,包括哺乳动物、两栖动物、鱼、植物、真菌和蓝细菌。不同生物金属硫蛋白理化特性和其氨基酸序列及中心片段的比较研究 ,对研究MT的结构和生物功能及生物的分子进化提供重要依据。哺乳动物MT研究较多 ,爬行动物鳖MT的研究尚属空白 ,本文报道鳖肝的金属硫蛋白。中华鳖(Pelodiscussinensis)分别经皮下注射ZnSO4 、CuSO4 和CdCl2 溶液诱导后 ,取乙醇沉淀的肝脏无细胞提取液再经SephadexG 5 0、DEAE SepharoseCL 6B及SephadexG 2 5凝胶过滤和离子交换柱层析分离 ,自鳖肝脏中分别获得Zn MT、Cu MT和Cd MT ,未经诱导的鳖肝脏中无MT。质谱和HPLC分析其分子量约为6 30 0dalton。根据氨基酸组成分析 ,鳖肝脏MT含 6 1个氨基酸残基 ,其中MT的典型氨基酸Cys含量占17%。Lys、Glu和Asp含量较高 ,而芳香族氨基酸和组氨酸含量极低。从紫外光谱特性分析 ,Zn MT、Cu MT、Cd MT紫外吸收肩分别在 2 2 0nm、 2 70nm和 2 5 0nm。表明确为鳖肝脏MT。从氨基酸残基数和分子量看 ,鳖肝脏MT与哺乳动物MT类似 ;而从氨基酸组成和结合金属离子的量看 ,又与低等生物蚯蚓及酵母菌的MT类似。

Metallothioneins ( MTs ) are a group of low molecular weight, metal-binding and cysteine rich proteins. MTs are widely distributed throughout living organisms and are fairly well identified in mammals, amphibians, fishes, plants, fungi, and cyanobacteria. To compare with the MT's- character, the high degree of conservation of amino acid sequence and the central segments in MTs from many sources will provide significant information for investigation of MT's structure and function or the molecular evolution. Thus far, there are no reports for MT isolated from reptiles. In the present paper, we report the presence of MT from turtle liver. Each extract of Chinese soft-shelled turtle (Pelodiscus sinensis) liver after induction with subcutaneous injection of ZnSO 4, CuSO 4 or CdCl 2 was separated by gel filtration chromatography Sephadex G-50 column and subsequent DEAE Sepharose CL-6B, than Sephadex G-25 for desalination, respectively. According to the results of mass spectrometry and HPLC filtration chromatography detection, the molecular weight of the MT is shown to be about 6 300 dalton. The MT containing 61 amino acid residues has a high cysteine content (17%), aromatic amino acids (tyrosine, phenylalanine), arginine and histidine are considerably low. Zn-MT, Cu-MT or Cd-MT has an ultraviolet absorption shoulder near 220 nm, 270 nm or 250 nm, respectively. In the present work we show that the properties of turtle liver MT are similar to the mammals in molecular weight and cysteine residues of MT. However, the other properties (including the amino acid composition of MT and the capability of binding metal ions) are similar to the MT from the earthworm and yeast. The characteristic of MT in turtle lies between the MT of mammals and lower living organisms. This result presents the distinguishing characteristics of biological evolution of MT in turtle.