Ricker R D, Kaji A
University of Pennsylvania, School of Medicine, Department of Microbiology, Philadelphia 19104-6076.
Anal Biochem. 1988 Nov 15;175(1):327-33. doi: 10.1016/0003-2697(88)90396-x.
Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2. Pure tRNAfmet was aminoacylated with [35S]methionine in the presence or absence of a formyl donor and was immediately fractionated on the column. Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other. The purified f[35S]-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro. Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.
使用己酸酐修饰的氨丙基硅烷-Hypersil 2进行混合模式高效液相色谱法，在30分钟内以基线分辨率将制备量的甲酰甲硫氨酰-tRNAfmet、甲硫氨酰-tRNAfmet和tRNAfmet彼此分离。纯tRNAfmet在有或没有甲酰供体的情况下用[35S]甲硫氨酸进行氨酰化，并立即在柱上进行分离。两种同工受体tRNA1fmet和tRNA2fmet以及氨酰-tRNA合成酶被清晰地彼此分离。纯化的f[35S]-甲硫氨酰-tRNA具有生物活性，因为在体外响应AUGUAA时，高达98%可与核糖体结合。通过终止因子的作用，甲酰甲硫氨酸从该复合物中释放出来，并且超过92%结合的甲酰甲硫氨酸被嘌呤霉素释放。
