The following reagents were obtained from Sigma-Aldrich: glucose, 1,3-propanediol, phenylboronic acid, cycloheximide, 23BD, acetoin, pyruvate, PEP, 6PG, G6P, F6P, Ru5P, R15P, ATP, dithiothreitol (DTT), NADH, NADPH, NADP+, phosphocreatine, RuBisCO, pyruvate kinase, lactate dehydrogenase, G6P dehydrogenase, 3PGA kinase, GAPDH and creatine kinase. U-13C glucose and 13C-NaHCO3 were obtained from Cambridge Isotope Laboratories. IPTG and chloramphenicol were obtained from Fischer Scientific. Gentamycin was purchased from Teknova. Spectinomycin was purchased from MP Biomedicals. Kanamycin was purchased from IBI Scientific. Phusion polymerase was purchased from New England Biolabs. All oligonucleotide synthesis and DNA sequencing were performed by Eurofins MWG Operon Inc.
G6p dehydrogenase
Manufactured by Merck Group
G6P dehydrogenase is an enzyme that catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-delta-lactone in the first step of the pentose phosphate pathway. It plays a key role in the generation of NADPH, which is essential for various cellular processes.
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Lab products found in correlation
Prism software, by GraphPad (2 mentions)
Hexokinase, by Merck Group (2 mentions)
Glucose 6 phosphate (g6p), by Merck Group (2 mentions)
Chloramphenicol, by Thermo Fisher Scientific (1 mentions)
Phosphocreatine, by Merck Group (1 mentions)
Acetoin, by Merck Group (1 mentions)
Gentamycin, by Teknova (1 mentions)
Phenylboronic acid, by Merck Group (1 mentions)
Spectinomycin, by MP Biomedicals (1 mentions)
Kanamycin, by IBI Scientific (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
U 13c glucose, by Cambridge Isotopes (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Rubisco, by Merck Group (1 mentions)
1 3 propanediol, by Merck Group (1 mentions)
13c nahco3, by Cambridge Isotopes (1 mentions)
Gapdh, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
7 protocols using g6p dehydrogenase
1
Metabolic Pathway Analysis with Isotopic Labeling
2
Enzymatic Assay for Antioxidant Capacity
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Nicotinamide adenine dinucleotide phosphate (NADPH), dithionite, glucose-6-phosphate (G6P), G6P dehydrogenase (G6PD), superoxide dismutase (SOD) and catalase were purchased from Sigma-Aldrich.
3
Measuring Hexokinase Activity in 832/13 Cells
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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.
4
Glucose Kinase Activity Measurement
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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.
5
Starch Content Quantification in Plants
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Starch content was assayed in seed and seedlings as previously described (Kanai et al. 2007 (link)) with minor modifications. Plant tissues were ground with a mortar and pestle in liquid nitrogen, and 5–10 mg of the resulting powder was washed twice with 1 ml of ethanol and twice with 1 ml of distilled water. The precipitate was suspended in 0.2 ml of distilled water and heated in a boiling water bath for 1 h, after which 100 µl of the solution was mixed with an equal volume of 50 mM sodium acetate buffer (pH 5.0). Glucoamylase (50 nkat) (Rhizopus niveus; Seikagaku Co.) and 5 nkat α-amylase (Sigma-Aldrich) were then added. The reaction mixture was incubated at 25°C for 1 h, then at 60°C for 1 h. The mixture was then centrifuged at 10,000 X g for 10 min, and 100 µl of the supernatant was mixed with 400 µl of a solution containing 60 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 2 mM NADP and 25 mM ATP. The amount of glucose in the solution was measured by the increase in absorbance at 340 nm after the addition of 1 µl each of hexokinase (2.8 nkat; Sigma-Aldrich) and G6P dehydrogenase (2.3 nkat; Sigma-Aldrich).
6
Enzymatic Activity Assay of ADP-glucose Pyrophosphorylase
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The assay was conducted at 30 °C in 100 mM HEPES NaOH buffer (pH 7.4) containing 3 mM 3-phosphoglycerate, 1.2 mM ADP Glucose, 3 mM sodium pyrophosphate, 5 mM MgCl2, 4 mM DTT, and 50 µl of crude enzyme extract in a reaction mixture volume of 250 µL. The reaction was allowed to continue for 20 minutes. The enzymes were inactivated thereafter by placing the mixture in a boiling water bath for 2 min. After addition of 350 µL of distilled water, the mixture was subjected to centrifugation at 15,000 g at 2 °C for 10 min. The supernatant (500 µL) was mixed with 0.15 mg NADP+. The enzymatic activity was recorded as the increase in absorbance at 340 nm after the addition of 1 µL each of phosphoglucomutase (0.4 units; Roche Diagnostics) and G6P dehydrogenase (0.5 units; Type XV, Sigma) with reference to Mohapatra et al.29 (link).
7
Quantification of Tissue Glycogen Content
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Samples of frozen tissues (30 to 90 mg) were hydrolyzed in 0.3 ml of 30% (wt/vol) KOH solution in a boiling water bath for 30 min. At 10 and 20 min of the incubation, tubes were vortexed to facilitate digestion. After cooling to room temperature, 0.1 ml of 1 M Na2SO4 and 0.8 ml of ethanol were added, the samples were then boiled again for 5 min to facilitate precipitation of glycogen and then centrifuged at 10,000 x g for 5 min. The glycogen pellet was dissolved in 0.2 ml of water, and two additional ethanol precipitations were performed. The final pellet was dried and dissolved in 0.2 ml of 0.3 mg/ml amyloglucosidase (Sigma Aldrich 11202332001) in 0.2 M sodium acetate buffer (pH 4.8) and incubated for 3 h at 40°C. The reaction mixture was diluted two-to fivefold with water. To determine the glucose concentration, 5 ml of the diluted sample was added to 0.2 ml of the glucose assay solution (0.3 M triethanolamine-KOH (pH 7.5), 1 mM ATP (Sigma Aldrich, A6559), 0.9 mM b-NADP (Sigma Aldrich, 10128031001), and 5 mg/ml of G-6P dehydrogenase (Sigma Aldrich, 10165875001)). The absorbance at 340 nm was determined before and after addition of 1 mg of hexokinase (Sigma Aldrich, 11426362001).
Glycogen content is expressed as micromoles of glucosyl units per gram (wet weight).
Glycogen content is expressed as micromoles of glucosyl units per gram (wet weight).
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