Fructose-1,6-bisphosphatase activity assay

To measure FbPase activity, cells were harvested, washed in PBS, and resuspended in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% Triton X-100, cOmplete Protease Inhibitor Cocktail [Roche]) at 2 x 10⁸ cells/ml. Following 20 min incubation at RT, cell extracts were centrifuged at 14,000 g, 16°C, 10 min, and supernatants were collected and kept on ice. The reaction mixture (20 mM Tris pH 7.8, 10 mM MgCl₂, 1 mM NADP, 1 μl glucose-6-phosphate isomerase [Sigma-Aldrich], 1 μl glucose-6-phosphate dehydrogenase [Sigma-Aldrich], 100 μl cell extract in H₂O) was incubated at 30°C for 5 min and 5 mM fructose 1,6-bisphosphate was added immediately prior to reading at 340 nm, 30°C with an UV-1601 spectrophotometer (Shimadzu).