| Assay Method Information | |
| | Inhibition of Tyrosine Decarboxylase In Vitro |
| Description: | Table 1: Tyrosine decarboxylase (tdc) was obtained by following a previously published literature procedure (Science, 14 Jun. 2019: Vol. 364, Issue 6445, eaau6323). Tdc (220 nM final concentration) was thawed on ice and then mixed with pyridoxal-5-phosphate (2.2 mM final concentration) in 200 mM sodium acetate buffer, pH 5.5 optionally containing 1 mM TCEP. To this mixture was added inhibitor at a final concentration of 1000, 333, 111, 37, 12, 4.1, 1.4, or 0 μM (final volume: 100 μL; inhibitor was 100-fold concentrated in a solution of DMSO, H2O, or DMSO:H2O (1/1 v/v)). The protein-inhibitor mixture was incubated at room temperature for 60 min. 6 μL of this mixture was then withdrawn from each solution and mixed with 54 μL of 10 mM levodopa in 200 mM sodium acetate buffer pH 5.5. The final concentration of the reaction was 22 nM tdc, 220 μM pyridoxal-5-phosphate, 9 mM levodopa in 200 mM sodium acetate buffer pH 5.5 with 0-100 μM inhibitor. The reaction was proceeded for 5 min at room temperature before quenching by addition of 540 μL acetonitrile containing 0.1 (v/v) formic acid supplemented with 200 nM tolbutamide as an internal standard. The reactions were centrifuged (3,000 g, 10 min), and then 100 μL of each supernatant was transferred to a fresh plate. 100 μL of acetonitrile containing 0.1% (v/v) formic acid supplemented with 200 nM tolbutamide was added. An external standard curve containing 0-150 μM dopamine was prepared in the exact same manner.Dopamine formed in each reaction was quantified by using an Agilent 6470 triple quadrupole mass spectrometer equipped with an Acquity UPLC. Mobile phase A consisted of H2O containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. Mobile phase B consisted of acetonitrile containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. 5 μL of each sample was injected onto a BEH Amide column (Waters Corporation, 2.1×50 mm, 1.7 μm). The gradient was set to: 100% mobile phase B at 0 min, decreasing linearly to 65% mobile phase B by 1.5 min, held constant at 65% mobile phase B until 2.5 min, ramped back up to 100% mobile phase B by 2.6 min, and held constant at 100% mobile phase B until 4.2 min. The flow rate was 0.6 mL/min. The dopamine was detected by using the mass spectrometer in multiple reaction monitoring (MRM) mode, quantifying the transition 154.1 to 137.0 m/z in positive mode. The fragmentor setting was 74, the collision energy was 9, and the cell accelerator voltage was 4, and the dwell time was 20. Tolbutamide was monitored using MRM and quantifying the transition of 271.1 to 91.0 m/z in positive mode. The fragmentor setting was 88, the collision energy was 37, and the cell accelerator voltage was 4, and the dwell time was 20. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |