Glucose 6 phosphate dehydrogenase

UDPG pyrophosphorylase (1) activity was analyzed using the spectrophotometric method using a coupled enzyme assay containing phosphoglucomutase (2) and glucose-6-phosphate dehydrogenase (3) according to the following equations:


The reaction mixture in the total volume of 1.0 mL contained 0.2 M HEPES–NaOH, pH 7.4, 6 mM MgCl₂, 2 mM sodium pyrophosphate, 10 mM UDPG, 0.4 mM NADP ⁺, 3 U glucose-6-phosphate dehydrogenase (Sigma-Aldrich, Saint Louis, MO, USA), 3 U phosphoglucomutase (Sigma-Aldrich) and 0.26 U/mg IAGlc synthase. The change of the NADPH/H⁺ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption coefficient 6220 M⁻¹ cm⁻¹ for NADH/H⁺.

To determine the levels of glucose, fructose, and sucrose, an enzymatic method was used as described previously [40 (link)]. An amount of 25 mg of lyophilized tomato fruits was mixed with 500 µL 80% (v/v) aqueous ethanol and incubated in a thermomixer at 1000 rpm at 80 °C for 1 h, followed by cooling on ice and centrifugation at 14,000× g at 4 °C for 5 min. The supernatant was collected and transferred to a new tube for evaporation to dryness (60–78 °C). The residue was dissolved in 250 µL water and subsequently used for sugar analysis in three technical replicates. Reactions were conducted in a 96-well plate with reaction mixtures each containing 100 µL diluted supernatant (7%) and 0.1 U glucose-6-phosphate dehydrogenase (Merck, Darmstadt, Germany) diluted in 100 µL buffer (100 mM Imidazole/HCl, pH 6.92, 10 mM MgCl₂, 2 mM ATP, 4 mM NAD). The reaction was performed in a microplate reader (Sunrise) and recorded at 340 nm wavelength for 3 min. Subsequently, 0.1 U hexokinase (Merck) was added to determine glucose level. The contents of fructose and sucrose were determined in the same way using 0.2 U phosphoglucose isomerase (Merck) and 1 U invertase (Merck), respectively. Levels were calculated using glucose, fructose, and sucrose as standards [23 (link)].

L-lactate concentration was determined according to a previously published protocol 30 . In brief, samples in 96-well microtiter plates were mixed with reaction buffer (0.1 M citric acid, 10 mg/mL BSA, 0.1% CaCl₂; pH 6.0) containing 0.25 units/mL lactate oxidase (Sigma-Aldrich, #L9795), 0.25 units/mL horseradish peroxidase (Sigma-Aldrich, #P8375) and 500 µM ABTS (Merck Millipore, #11204521001). After incubation for 1 h at room temperature, absorbance at 405 nm was determined.
Alternatively, lactate concentration in cell culture supernatants or mouse brain tissue homogenates was determined using the Colorimetric L-lactate assay kit (Merck Millipore, #MAK329) according to the supplier's protocol.
Glucose concentration in cell culture supernatants was determined according to a previously published protocol 31 (link). In brief, samples in 96-well microtiter plates were mixed with reaction buffer (177 mM Tris HCl, 10 mM MgCl₂; pH 7.8) containing 5 mM ATP (Merck Millipore, #10127523001), 2.5 mM nicotinamide adenine dinucleotide phosphate (NADP; Carl Roth, Karlsruhe, Germany, #AE13.1), 3 U/mL hexokinase (Merck Millipore, #11426362001), 3.4 U/mL glucose-6-phosphate dehydrogenase (Merck Millipore, #10165875001). After incubation for 1 h at room temperature, absorbance at 340 nm was determined.