Glycerophosphate dehydrogenase

Enzyme assays were performed using protocols described in Methods in Enzymology (Volume 9) [75 -77 ]. Briefly, the cell pellets were suspended in cold 50 mM potassium phosphate buffer containing 10% glycerol, 10 mM DTT and a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO). Suspensions were sonicated followed by centrifugation at 12,000g and the supernatants were collected for enzyme assays. Extracts were resuspended in 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM Sodium pyruvate and 0.13 mM NADH for LDH (lactate dehydrogenase) assays, or 20 mM Tris-HCl buffer (pH 7.6) containing 15 mM glucose, 20 mM MgCl₂, 0.13 mM NADP, 0.01mM EDTA, 1 mM ATP and 0.2 units of glucose-6-phosphate dehydrogenase (Sigma-Aldrich, St. Louis, MO) for the hexokinase assay. An aldolase assay was performed by resuspending extracts in a buffer containing 50 mM glycylglycine (pH 7.5) containing 0.2 mM NADH, 10 mM FDP and 15 units of glycerophosphate dehydrogenase (Sigma-Aldrich, St Louis, MO). Changes in optical density were measured at room temperature at a wavelength of 340 nm and the units of activity were reported as the changes in optical density per minute per 1 mg protein, as in [75 -77 ]. Total protein content per extract was measured using Coomassie brilliant blue (Sigma-Aldrich, St. Louis, MO).