HK activity assay

The activity of hexokinases (HKs) was determined by spectrophotometric measurement of the formation of β-nicotinamide adenine dinucleotide (NADH) that is coupled with the oxidation of G6P, the product of glucose phosphorylation by HK (Polakis and Wilson, 1982; Al jamal, 2005). The coupled assay produces 1 μmol of NADH per μmol of D-glucose phosphorylated.

Neuroblastoma cells were lysed as described before (Edry Botzer et al, 2011), diluted in lysis buffer to a volume of 70 μl and added in duplicates to a 96-well plate on ice. Reaction buffer (20 mM Hepes pH=7.2, 5 mM MgCl₂, 5 mM KCl and 10 mM glucose, freshly supplemented with 1 U ml⁻¹ PK, 1 U ml⁻¹ LDH, 1 mM ATP, 1 mM PEP and 0.18 mg ml⁻¹ NADH) was added to the wells in a volume of 200 μl and the plate was immediately read using an automated microplate reader (SpectraMax 190; Molecular Devices Corp., Sunnyvale, CA, USA) at 25 °C, OD₃₄₀. Measurements were taken every 15 s for 25 min. The HK activity was calculated as follows: ΔOD/min (Vmax)−ΔOD/min blank × 1000 (for 1000 μl) × dilution factor/6.22 (mM extinction coefficient of NADH).