Aminoacyl Transfer Ribonucleic Acid Synthesis in Toluene-treated Liver Cells

SUMMARY Aminoacyl-tRNA synthetases were studied in suspensions of individual liver cells made permeable to tRNA by treat- ment with toluene. Centrifugation of the toluene-treated cells revealed that about 90% of the aminoacyl-tRNA syn- thetase activity remained associated with the cell pellet. If the toluene-treated cells were preincubated under assay conditions and then centrifuged, about ‘75% of the amino- acyl-tRNA synthetase activity remained associated with the cell pellet suggesting that aminoacylation occurred within the cell. Aminoacyl-tRNA synthesis in this system was dependent on exogenous tRNA, ATP, and Mg2+. Enzyme activity remained linear with time through about 10 min of incubation and a IO-fold increase in cell concentration. The product of the reaction was exclusively aminoacyl-tRNA. This system should prove useful for study of macromolecular synthesis in mammalian cells. Toluene treatment of bacteria has been used for a number of years to make such cells permeable to exogenous substrates (1). This procedure has received considerable attention recently in studies of DNA replication since the toluene-treated cells appear to remain relatively intact and synthesize DNA normally (2). Generally, toluene treatment has been used to render bacteria permeable to low molecular weight compounds, although it is known that pancreatic DNase can enter treated cells (2), and that the presence of Triton X-100 also makes such cells permea- ble to large proteins (3). To our knowledge, toluene treatment has not been used to study macromolecular syntheses in cells of mammalian origin. Several years ago our laboratory reported the isolation from rat liver of a high molecular weight complex containing all the aminoacyl-tRNA synthetases and a large proportion of the cellu- lar tRNA (4). In a continuation of these studies we have at- tempted to examine aminoacyl-tRNA synthetases under condi- tions which would lead to minimal disruption of the complex. For this purpose we have been working with suspensions of in- dividual liver cells made permeable by treatment with toluene. In this communication we report that aminoacyl-tRNA synthe- tases can be studied within such toluene-treated liver cells, and that these cells are permeable to exogenous tRNA. Livers were obtained from male Long-Evans rats (125 to 150 g) fed ad