OASTL activity assay monitoring cysteine biosynthesis
200 mg Arabidopsis wild-type Col-0 leaves were homogenized in liquid nitrogen. 0.5 mL extraction buffer (50 mM HEPES-KOH, pH 7.5; 10 mM KCl; 1 mM EDTA; 1 mM EGTA; 30 mM DTT; 0.5 mM PMSF and 10% (v/v) glycerol) was added and mixed at 4 °C for 10 min with frequent shaking. After centrifugation at 16,000 g for 10 min, supernatant was collected. Protein concentration was measured with ROTI®Quant (Carl-Roth, Germany) following manufacturer’s instruction.
The OASTL activity assay was carried out in a volume of 0.1 mL containing 100 mM HEPES-KOH pH 7.5; 5 mM DTT; 10 mM OAS and 10 mM Na2S or 4 mM TMTM. The reaction was initiated by the addition of OAS, and was incubated for 10 min at 25 °C. Termination of the reaction was done by adding 50 μL of 20% (w/v) trichloroacetic acid followed by centrifugation at 12500 g.
100 µL of the supernatant was transferred to a new tube and incubated in 200 µL of 134 mM Tris-HCl, pH 8.0 and 1 mM DTT at room temperature for 30 min. The reduced sample was mixed with 200 µL acetic acid and 200 µL ninhydrin reagent (250 mg ninhydrin dissolved in 6 mL acetic acid and 4 mL concentrated HCl). The tube was incubated at 90 °C for 10 min, then cooled rapidly on ice for 2 min. The sample was diluted with 95% ethanol and measured at 560 nm to quantify the synthesized cysteine.