Aldolase

PFK and GK ADP-dependent activities were determined as previously reported (Kengen et al., 1994 (link)). Briefly, the GK activity was determined by following the reduction of NAD⁺ at 340 nm in a coupled assay with 10–13 units of G6PDH from Leuconostoc mesenteroides, which was heterologously expressed in E. coli and purified in our laboratory. The assay also contained 25 mM HEPES pH 7.8 and 0.5 mM NAD⁺. The PFK-ADP activity was measured by following the oxidation of NADH at 340 nm in a coupled assay with the following auxiliary enzymes: α-glycerol-3-phosphate dehydrogenase (5 units), triosephosphate isomerase (50 units) and aldolase (1.3 units) all from rabbit muscle (Sigma-Aldrich), along with 25 mM PIPES pH 6.5 and 0.2 mM NADH. All enzyme activity determinations were performed at 40°C and the specific activity was calculated from the initial velocity data. The unit of enzyme activity (U) was defined as the conversion of 1 μmol of substrate per minute. Kinetic parameters for the PFK and GK-ADP activities were determined varying the concentration of one substrate at a fixed and saturating concentration of the co-substrate. The initial velocities determined were adjusted either to the Michaelis-Menten or substrate inhibition equations by non-linear regression.

Lyophilised proteins BSA, lysozyme, and lactoferrin were purchased from Sigma–Aldrich and dissolved in 20 mM HEPES, 150 mM NaCl at pH 7.4. An ammonium sulphate solution of aldolase was purchased from Sigma and dissolved in 20 mM HEPES, 150 mM NaCl at the required pH, and ammonium sulphate was removed using an Amicon filter.

The protein samples were loaded onto the column (Sephacryl S100 HR) and separated into fractions at a flow rate of 2.5 ml/min (20°C). The column was pre-calibrated with the following proteins (Sigma—Aldrich): thyroglobulin (660 kDa), catalase (440 kDa), aldolase (158 kDa), BSA (67 kDa), γ-crystallin (20 kDa). The relative error for protein mass determination was 4%.

Antibodies against the following proteins/epitopes were used for immunoblot with the sources, catalog numbers, and dilutions indicated: Actin (Sigma-Aldrich, St Louis, MO; A2066, 1:5000), Flag (Sigma-Aldrich, F3165, 1:10,000), p73 (Bethyl Laboratories, Montgomery, TX; A300–126A, 1:1000), G6PD (Sigma-Aldrich, HPA000834, 1:1000), p21 (BD Bioscience, San Jose, CA; 556431, 1:1000), PFKL (Santa Cruz Biotechnology, Dallas, TX; sc-292523, 1:1000) (Abcam, Cambridge, UK; ab181064, 1:2000), p53 (Santa Cruz sc-126HRP, 1:2000), PFKM (R&D Systems, Minneapolis, MN; MAB7687, 1:1000), PFKP (Cell Signaling Technology, Danvers, MA; 8164S, 1:1000), 6PGD (Abgent, San Diego, CA; AP5448c, 1:1000) (Santa Cruz, sc-39877, 1:500), and V5 (EASYBIO, BE2033, 1:2000).
Etoposide (ETO) was purchased from Selleck. The following reagents were purchased from Sigma-Aldrich: ATP, AMP, NAD⁺, NADH, NADP⁺, NADPH, doxorubicin (DOX), crystal violet (CV), 2′,7′-Dichlorofluorescin diacetate (DCF), citrate, fructose 6-phosphate (F6P), triose phosphate isomerase (TPI), aldolase (ALDO), and α-glycerophosphate dehydrogenase (GAPDH).