论文部分内容阅读
本文报道了用一种高灵敏度的方法测定人工合成的酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))的生物活力。tRNA_y~(Ala)在大鼠肝氨酰基tRNA合成酶的催化下接受丙氨酸后,用操作简便而回收率较高的酒精沉淀法回收氨酰化产物,最后,在兔网织红细胞裂解液无细胞蛋白合成体系中,测定氨酰化产物中的丙氨酸转移到蛋白质中去的能力——参入活力。这方法不仅可以测定分离纯化的tRNA_y~(Ala)的活力,而且也可以测定经T_4RNA连接酶连接两个半分子后的反应混合物中产物tRNA_y~(Ala)的活力。利用这方法,已成功地测定了微至5—7 pmoles的人工全合成tRNA_y~(Ala)的接受活力和参入活力两组数据。测定结果表明,全合成tRNA_y~(Ala)的接受活力是天然分子的51.6—65.6％,是经拆合的天然分子的91.3—106.0％。其氨酰化产物中的[~3H]-Ala在兔网织红细胞裂解液中的利用率为61.6—63.1％,是天然分子的90.6—91.7％,是经拆合的天然分子的97.2—115.8％。
In this paper, a highly sensitive method was reported for the determination of the biological activity of synthetic yeast alanine-transfected RNA (tRNA_yAla). After being alanine catalyzed by rat hepatic aminoacyl tRNA synthetase, tRNA_y ~ (Ala) was used to recover the aminoacylated product by an ethanol precipitation method with high recovery rate and simple operation. Finally, in the reticulocyte lysate In a cell-free protein synthesis system, the ability of alanine in an aminoacylated product to be transferred to a protein is determined-inactivation. This method can not only determine the activity of isolated and purified tRNA_y ~ (Ala), but also determine the activity of tRNA_y ~ (Ala) in the reaction mixture after two half molecules are ligated by T_4 RNA ligase. Using this method, two sets of data of the viability and the viability of artificially synthesized tRNA_yAla from as little as 5-7 pmoles have been successfully measured. The results showed that the total viability of tRNA_y ~ (Ala) was 51.6-65.6% of natural molecules, which was 91.3-106.0% of natural molecules disassembled. The utilization ratio of [~ 3H] -Ala in rabbit lysate was 61.6-63.1%, which was 90.6-91.7% of the native molecule and 97.2-115.8 of the native molecule disassembled %.