Swiftii

For the determination of specific enzyme activity, cells were grown until stationary phase, washed once with 25 ml water, and resuspended in sodium phosphate buffer (100 mM, pH 7.0). Cell extract was made with glass beads (0.5 mm) and precellys 24 bead beater with a cryolys cooling unit (Bertin technologies, Aix-en-Provence Cedex, France). Total protein amount was determined by Bradford with bovine serum albumin (Fermentas UAB, Vilnius, Lithuania) as standard [37 (link)]. Reductase activity was assayed by following the oxidation of NAD (P) H at 340 nm using Ultrospec 2100pro spectrophotometer (Amersham Biosciences, Sweden). Data were collected using the software SWIFTII (Amersham Biosciences, Sweden). Samples were diluted so that the absorbance decreased linearly during 5 min. One unit of activity corresponds to 1 μmol NAD (P) H consumed per minute at 30°C. The assay contained sodium phosphate buffer (100 mM, pH 7.0), NAD (P) H (200 μM), ACP (10 mM) and cell extract.

Cell extract was prepared with Yeast Protein Extraction Reagent (Thermo Scientific, Pierce, Rockford, USA) according to the instructions provided by the manufacturer. The total protein concentration in cell extracts was determined using the Bradford method [34 (link)] with bovine serum albumin (BSA) as standard. SADH activity measurements were performed as described previously [35 (link)]. The activity is based on measuring the oxidation of NADH at 340 nm with an Ultrospec 2100 pro spectrophotometer (GE Healthcare Life Sciences, Sweden). The data were collected with the software program SWIFTII (Amersham Biosciences, Sweden). Cell extracts were diluted until the decrease in absorbance was linear for 5 min, at which point the activity could be calculated from the slope. One unit of activity corresponds to 1 µmol NADH consumed per minute at 25 °C. The assay contained sodium phosphate buffer (50 mM, pH 7), acetophenone (10 mM), NADH (0.2 mM), and cell extract (1-30 mg/l total protein).