Glucose 6 phosphate assay kit

The animals were fasted overnight for 18 h, then injected intraperitoneally with 3 g/kg glucose. After 1 h, the pancreas was removed, and Glucose-6-Phosphate (G6P) was measured using Glucose-6-Phosphate Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), per the manufacturer’s instructions.

The activity of HAPS_0849 was confirmed with a Glucose-6-Phosphate Assay Kit (Sigma, MAK014) according to the manufacturer’s manual, with some modifications. Briefly, 10 µL of 1 mM G1P was added to a 96-well plate, and then G6P assay buffer was added to each well to bring the volume to 50 µL. Another 50-µL reaction mix containing 45 µL of G6P Assay Buffer, 2 µL of G6P Enzyme Mix, 2 µL of G6P Substrate Mix and 1 µL of protein sample or phosphate buffered saline (PBS) as a negative control was prepared. Next, 50 µL of the reaction mix was added to 50 µL of the 1 mM G1P solution described above and mixed well by pipetting. The reaction was incubated for 30 min at room temperature, and then the absorbance was measured at 450 nm (A450). To eliminate the effect of background NADH or NADPH, a blank sample was prepared for each sample by omitting the glucose-6-phosphate enzyme mix. The blank readings were subtracted from the sample readings. Activity was calculated as U (units) = (A_s − A_b)/(T₁ − T₀) × df (where A_s, A450 of sample after 30 min; A_b, A450 of sample without G6P enzyme mix as a blank; T₁, time reaction was stopped; T₀, time reaction began; df, dilution factor of protein). Phosphoglucomutase from rabbit muscle (Sigma Aldrich, USA) was used as positive control.