使用C6修饰的氨丙基硅烷化高效硅胶在混合模式高效液相色谱上分离甲酰甲硫氨酰转运RNA、甲硫氨酰转运RNA和甲硫氨酰起始转运RNA。

Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil.

作者信息

Ricker R D, Kaji A

机构信息

University of Pennsylvania, School of Medicine, Department of Microbiology, Philadelphia 19104-6076.

出版信息

Anal Biochem. 1988 Nov 15;175(1):327-33. doi: 10.1016/0003-2697(88)90396-x.

DOI:10.1016/0003-2697(88)90396-x

PMID:2469355

Abstract

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.

摘要

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。