Measuring Hexokinase Activity in 832/13 Cells

To evaluate the effect of GKRP overexpression over GK activity, 832/13 cells were transduced with 5 × 10⁷ ifu/mL AdGKRP or Ad-Control and incubated for 72 h. After that, cells were lysed by ultrasound and promptly HK activity was determined as previously described with slight modifications (Salgado et al., 2014 (link)). Briefly, a 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, 1 mM ATP, 0.5 mM NADP⁺, 1–30 mM 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 velocity reaction for each absorbance, we made a calibration curve with G-6P as substrate.