Reversed phase chromatographic analysis of Nostoc and Euglena isoleucyl-tRNAs aminoacylated in vitro in homologous and heterologous systems

Abstract

High pressure reversed-phase chromatography (RPC-5) of tRNA from the blue-green alga, Nostoc, acylated with [ 14C]Ile in a homologous system indicated the presence of 2 major isoleucyl-tRNA species. Analysis of Nostoc tRNA acylated in a heterologous system (enzyme from green or aplastidic Euglena) showed 2 isoleucyl-tRNA species present, one of which eluted from the column at a different salt molarity than that required for elution of either of the isoleucyl-tRNAs seen in the homologous Nostoc system. Omission of unlabeled valine from reactions in the heterologous system resulted in the appearance of a third elution peak, presumably Ile mischarged to tRNA Val. In the homologous Euglena system, if the source of tRNA, enzyme or both was from aplastidic cells then 2 isoleucyl-tRNA species were noted, with the peak of the earlier-eluting species being four times larger in size than that of the later-eluting species. If both enzyme and tRNA came from green Euglena then the 2 peaks of isoleucyl-tRNA seen were of equal size. We think that the earlier-eluting peak represents the cytosol species of isoleucyl-tRNA and suggest that the mitochondrial and chloroplast species coelute from the column at the higher salt molarity. Analysis of tRNA from green Euglena acylated by Nostoc enzyme revealed 2 isoleucyl-tRNA species with the elution position of neither corresponding to that attributed to the Euglena cytosol species. Under conditions tested Nostoc enzyme preparations could not charge Ile to tRNA from aplastidic Euglena. Differences noted in the elution profiles of isoleucyl-tRNA species when analyses of heterologous systems were compared to those of homologous systems may reflect modification as well as aminoacylation activities specific to each enzyme source.