β nicotinamide adenine dinucleotide reduced disodium salt nadh

MPST activity was determined by the method of Valentine and Frankelfeld [46 (link)], according to the procedure described by Wróbel et al. [47 (link)]. Firstly, the incubation mixture was composed of 250 µL 0.12 M sodium phosphate buffer (pH 8.0), 50 µL 0.5 M sodium sulfite (Sigma-Aldrich), 50 µL 0.15 M DL-Dithiothreitol (DTT, Sigma-Aldrich), 50 µL distilled water, 50 µL 0.1 M sodium salt of 3-mercaptopyruvate acid (Santa Cruz Biotechnology, Dallas, TX, USA), and 50 µL homogenate. The total volume of 500 µL was incubated for 15 min. Then, 250 µL of 1.2 M perchloric acid (PCA, POCh S.A., Gliwice, Poland) was used to stop the reaction. The samples were centrifuged at 1600× g for 5 min. A total of 100 µL of the supernatant was transferred to a mixture containing 1200 µL 0.12 M sodium phosphate buffer (pH 8.0), 100 µL 0.1 M N-ethylmaleimide (Sigma-Aldrich), and 50 µL 5 mg/mL β-Nicotinamide adenine dinucleotide reduced disodium salt (NADH, Sigma-Aldrich). After equilibration at 37 °C, 2.5 µL (7 IU) L-lactic dehydrogenase (Sigma-Aldrich) was added and the absorbance was measured at 340 nm. The enzymatic activity was calculated as µmoles of pyruvate produced during the 1 min incubation at 37 °C per 1 mg of protein.