Glucose Kinase Activity Measurement

To evaluate the effect of GKRP overexpression on GK activity, 832/13 cells were transduced with 5 x 10⁷ IFU/mL each adenovirus and incubated for 72 h. After that, cells were lysed by ultrasound, and HK activity was promptly determined as previously described with slight modifications 50 (link). Briefly, a glucose 6-phosphatase (G-6P) dehydrogenase-coupled reaction was used, and the activity was followed by measuring the increase in absorbance at 340 nm after 5 min incubation at 37°C. The reaction mixture consisted of 200mM Tris-HCl buffer (pH 7.5), 2mM MgCl₂, 1mM DTT, 1mM ATP, 0.5mM NADPH, 1-30mM glucose, and 1 U/mL of G-6P dehydrogenase (Sigma-Aldrich). For specific activity determination, we used 0.5 mg/mL of total protein in the reaction mixture, and the Prism software was used for data analysis (GraphPad, Inc.). To determine the reaction velocity for each absorbance, we made a calibration curve with G-6P as a substrate.