Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption.

Reversed-phase chromatography separates lysyl-tRNA from mammalian tissues into five isoaccepting species. The major peaks are designated lysyl-tRNA₂ and lysyl-tRNA₅. In ribosomal binding experiments lysyl-tRNA₂ binds exclusively for AAG, whereas lysyl-tRNA₅ codes preferentially for AAA. The results presented here show that the lysyl-tRNA₅ peak in liver tissue is a mixture of two different lysyl-tRNAs which we have designated as lysyl-tRNA₅ and lysyl-tRNA_5B. These two lysyl-tRNAs were distinguished by the fact that lysyl-tRNA_5B could still accept lysine after iodine oxidation, whereas lysyl-tRNA₅ could not. Lysyl-tRNA_5B, however, was modified by the iodine treatment because it eluted at a different position during RPC-5 chromatography. Similar results were obtained when cyanogen bromide was used in place of iodine as the modifying reagent. It was also shown that iodine oxidation of lysyl-tRNA_5B caused a loss of the ability of this tRNA to bind to ribosomes in response to both ApApA and ApApG. Reversal of the effects of iodine by incubation with sodium thiosulfate restored some of the ribosomal binding activity. Under these conditions lysyl-tRNA_5B bound in response to ApApG, but not to ApApA. Based upon these data, lysyl-tRNA_5B appears to be a sulfur-containing lysyl-tRNA which codes for AAG exclusively. It is postulated that this tRNA may contain a 2-thiocytidine in the anticodon loop.