Trp Uptake Kinetics in HeLa Cells

Trp uptake experiments were performed as previously reported [19 (link),38 (link)]. In detail, the HeLa cells were extensively washed (3×) in PBS (pH 7.4), and then resuspended in uptake assay buffer (PBS plus 0.3 mM MgCl₂) to give 1 × 10⁶ cells/mL. [³H]Trp (150 nM) was added to a 0.3 mL aliquot of cell suspension plus or minus 500 nM TrpRS protein. Cellular uptake of Trp was then monitored at 25 °C over a series of timepoints (0, 1, 2, 3, and 4 min). Specifically, 50 μL samples were subjected to rapid vacuum filtration through a glass microfiber filter (GE Healthcare Life Sciences) with a particle retention of 1.2 μm. Filters were washed (5×) with 5 mL PBS, air-dried, and analyzed on a scintillation counter (PerkinElmer, Waltham, MA, USA). Trp uptake increased over time in a linear fashion. Uptake was found to be linear, and the initial rate was calculated (fmol min⁻¹).