Leucyl-tRNA synthetases in Chinese hamster ovay cells : functional studies on wild-type cells
Isoye, Lynn Akiko
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A number of studies on aminoacyl-tRNA synthetase (aaRS) complexes from Chinese hamster ovary (CHO) wild-type (WT) and temperature-sensitive mutants, indirectly support a generalized model for the true subcellular role of the aaRS complexes in the initiation of protein synthesis in mammalian cells. This model proposes that the high molecular weight (HMW) complexes utilize extracellular amino acids immediately upon transport into the cell before equilibrating with the intracellular amino acid pool which is exclusively utilized by the low molecular weight aaRS form. The WT CHO cells were selected for this study to determine the functional significance of the leucyl-tRNA synthetase (LeuRS) complexes. For controls, cells were processed through an amino acid transport assay where L-leucine was depleted with α(-MEM-[-leucine] media, re-loaded for uptake with L-leucine for a specific time, lysed and placed directly onto Sephadex G-75 microcentrifuge desalting columns. Protocols for the above techniques were slightly modified so as not to disrupt the normal LeuRS complex profiles seen on sucrose density gradients. LeuRS sedimentation profiles for each procedure were comparable to that of the normal LeuRS forms, although with each progressive procedure a slight reduction in activity of each LeuRS form was seen. These techniques may be too harsh where the low LeuRS activity is possibly due to breakdown of the complexes. Eventually, for the experimental cell sample, L-[¹⁴C]-leucine and L-[³H]-leucine, representing the intracellular and the extracellular pools, respectively, were added for uptake and the microcentrifuge desalting columns were used to remove excess unbound labelled L-leucine. A tRNA assay was further utilized to compare the dual-labelled cell fractions treated with tRNA (+) and without tRNA (-) to determine the relative levels of the labelled L-leucine incorporated into the leu-LeuRS-AMP intermediate complex via the high and low molecular weight LeuRS forms. There were no significant variations among the L-[¹⁴C/³H]-leu- LeuRS-AMP mean ratio and standard error of the mean for the high and low molecular weight LeuRS forms. This preliminary study indicates that the many variables in these protocols hinder the true measure of aminoacyl adenylate complexes and must be overcome to successfully determine the functional aaRS model.