Glucose 6 phosphate (g6p)

In this study, in vitro metabolic activation via the addition of the rat liver S9 fraction (Sigma Aldrich, Saint Louis, USA) was used to metabolize the examined Japanese herbal medicines. Metabolic activation was induced using an S9 mixture containing 0.5 mg/ml of the S9 fraction in 100 mM potassium phosphate buffer (pH 7.4), 3.3 mM MgCl₂ (Wako Chemical, Japan), and an NADPH-generating system [1.3 mM NADP (Sigma Aldrich, USA), 3.3 mM glucose 6-phosphate (Oriental Yeast, Tokyo, Japan), and 0.4 units/ml of glucose-6-phosate dehydrogenase (Oriental Yeast, Tokyo, Japan)]. Each 200-μl sample of Japanese herbal medicine dissolved in DMSO was incubated with 800 μl of the S9 mixture at 37°C for 1 h or with 800 μl of the negative mixture, which was composed of the abovementioned S9 mixture minus the S9 fraction, as a negative control. The metabolized Japanese herbal medicines were stored at − 80°C until use.

The UV-visible absorption spectra were measured with a double beam spectrophotometer (V-560, JASCO, Tokyo, Japan) at room temperature (20–25 °C). Protein subunit and substrate concentrations were determined using the extinction coefficients for Y50G and Y50S BsFNRs (ε₄₅₇ = 12.7 mM⁻¹ cm⁻¹ [29 (link)]), Y50W BsFNR (ε₄₅₈ = 11.8 mM⁻¹ cm⁻¹ [29 (link)]), BsFd (ε₃₉₀ = 16.0 mM⁻¹ cm⁻¹ [39 (link)]), and NADPH (ε₃₄₀ = 6.2 mM⁻¹ cm⁻¹). NADP⁺ concentration was determined in reduced NADPH form after incubation for several minutes in the presence of 10 mM glucose-6-phosphate (Oriental yeast Co., Ltd.) and 5 U/mL glucose-6-phasphate dehydrogenase (from Leuconostoc mesenteroides, Worthington Biochemicals Co., Ltd., Lakewood, NJ, USA).
The representation of the protein 3D-structure was performed with BIOVIA Discovery Studio Visualizer (Ver. 21.1, Dassault Systèms, Vélizy-Villacoublay, France).