用质粒作底物测定核糖体失活蛋白酶活性的新方法

植物核糖体失活蛋白(ribosome-inactivating protein,RIP)是一类能作用于核糖体最大RNA的独特蛋白质.它是研究蛋白质生物合成中核糖体RNA结构与功能的有力工具.利用RIP能在DNA中脱去一些腺嘌呤碱基使超螺旋DNA解旋的特点,分别以常用的质粒PUC18、PUC19和PBR322 DNA为底物,建立了测定RIP酶活性的一种新方法,其灵敏度是50ng(天花粉蛋白)和5ng(还原型的辛纳毒蛋白),酶催化反应的时间是60min.这个新方法具有方便、快捷、灵敏的特点,避免了常用方法中制备核糖体、提取RNA的仪器和技术条件的限制,检测的时间由原来的几天缩短到约120min,大大地降低了检测的费用,为广泛和深入地研究RIP提供了有利的条件.

A Novel Method for Assaying Enzymatic Activity of Ribosome-inactivating Protein Using Plasmid DNA as Substrate

A novel method for assaying the enzymatic activity of ribosome-inactivating proteins(RIPs) has been developed.The principle of the method is based on that RIP can remove some adenine bases from double-stranded supercoiled DNA molecules,subsequently,the deadenylated DNA was cleaved into nicked and linear form.After treatment with acidic aniline,the deadenylated DNA was degraded into many small fragments,and run out of the gel.The enzymatic activities of two RIPs(trichosanthin and cinnamomin) were tested using this method,the limit of sensitivity is about 50 ng(trichosanthin) and 5 ng(reduced cinnamomin) .It should be emphasized that the merit of this method is to avoid the preparation of ribosome.