作　　者： ; ;

机构地区： 北京大学生命科学学院

出　　处： 《中国生物化学与分子生物学报》 1999年第5期772-778,共7页

摘　　要： 金属硫蛋白具有α和β两个独立的结构域,它们结构不同,并能独立的行使功能。为了进一步研究这两个结构域之间的区别,分别采用镉和铜重组金属硫蛋白并继以枯草杆菌蛋白酶水解的方法制备α和β结构域,以及利用pGEX-4T-1这种融合表达载体表达α和β结构域。所得产物经凝胶过滤层析分离纯化后,进行了氨基酸组成,巯基和金属含量以及分子量测定,以上性质均与天然的金属硫蛋白α和β结构域相同。然后利用紫外吸收光谱和圆二色吸收光谱来研究它们的巯基金属簇结构,从UV和CD图谱可以看出,通过蛋白水解和基因表达制备的α和β结构域都具有独立的镉硫金属簇结构,但β结构域的镉硫金属簇不如α结构域紧密,其在254nm的吸收峰不象α结构域那么明显。利用DTNB的竞争反应测定了α和β结构域对镉和锌的结合力,实验结果表明,α结构域倾向于结合Cd2+,β结构域倾向于结合Zn2+。以上研究对于进一步了解α和β结构域的生理功能和分子进化提供了有利的证据。 Metallothionein folds into two separate domains that exhibit different structure and function independently In order to study further on the difference between these two domains,the separate α and β domains were prepared from proteolysis and gene expression respectively First,cadmium and copper reconstitution followed by hydrolysis with subtilisin was used to obtain α and β domains Second,pGEX 4T 1 vector was chosen to express α and β domains as the C terminal of GST protein After purified by gel filtration,the product was identified by molecule weight with mass spectroscopy,amino acid composition,metal and sulphydryl content All these properties were the same as the natural α and β domains of MT Then UV spectroscopy and CD spectroscopy were used to prove the metal cluster structure,from the UV and CD spectra,α and β domains prepared by proteolysis and gene expression were found to exhibit the independent Cd thiolate cluster structure,but the cluster of β domain was more relaxed than that of α domain The determination of metal binding ability with DTNB reaction showed that Cd prefered to bind with α domain while Zn with β domain,which gave some evidence to the physiological function and the evolution of two domains of MT

关 键 词： 金属硫蛋白 结构 功能 结构域 结构域

领　　域： [生物学] [生物学]