Tissue F2,6-BP concentration was determined as previously described (Van Schaftingen, Lederer, Bartrons, & Hers, 1982 (link)). Briefly, samples of tissues were weighed and homogenized in NaOH (0.05 M). The resulting mixture was heated for 20 min at 80°C. After cooling, the samples were neutralized with 1M acetic acid in the presence of 20 mM Hepes, and then centrifuged. Samples were incubated at 37 °C for 5 min in the following assay mixture: 50 mM Tris, 5 mM Mg2+, 1 mM fructose-6-phosphate (Sigma #F3627), 0.15 mM NADH (Sigma #N4505), excessive PPi-dependent PFK-1 (enriched from potato tubers), 0.2U/mL aldolase (Sigma #A2714), 8U/mL triosephosphate isomerase (Sigma #T2507) and 1U/mL glycerol-3-phosphate dehydrogenase (Sigma #10127752001). After the 5 min pre-incubation time, 0.5 mM pyrophosphate was added to start the reaction, and the rate of change in OD340 nm every 30 seconds was followed for 5 min in a Bio-Rad xMark microplate spectrophotometer (Bio-Rad). Data are expressed as the fold change compared to the WT controls.
Glycerol 3 phosphate dehydrogenase
Manufactured by Merck Group
Sourced in Japan, United States
Glycerol-3-phosphate dehydrogenase is an enzyme that catalyzes the reversible oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. It is involved in glycolysis and gluconeogenesis pathways.
Automatically generated - may contain errors
Lab products found in correlation
Aldolase, by Merck Group (5 mentions)
Triosephosphate isomerase, by Merck Group (5 mentions)
F2 6bp, by Merck Group (2 mentions)
Xmark microplate spectrophotometer, by Bio-Rad (1 mentions)
Saccharomyces cerevisiae, by Merck Group (1 mentions)
Varioskan microtiter plate reader, by Thermo Fisher Scientific (1 mentions)
2 deoxy d ribose, by Merck Group (1 mentions)
Glutamate dehydrogenase, by Merck Group (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
L ala, by Fujifilm (1 mentions)
Ethanol, by Fujifilm (1 mentions)
L ser, by Fujifilm (1 mentions)
L threonine, by Fujifilm (1 mentions)
L pro, by Merck Group (1 mentions)
L lysine hydrochloride, by Merck Group (1 mentions)
8 protocols using glycerol 3 phosphate dehydrogenase
1
Quantifying F2,6-BP in Tissue Samples
2
Quantification of Intracellular F2,6P2 Levels
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The intracellular F2,6P2 concentration was measured as previously described.26 (link) Briefly, cell extracts were incubated in a mixture supplemented with 50 mmol/L Tris/HCl, 0.15 mmol/L NADH, 5 mmol/L MgC12, 5 kilounit/liter triose-phosphate isomerase, 0.45 kilounit/liter aldolase, 1 mmol/L F6P (fructose 6-phosphate), 10 units/liter PPi (pyrophosphate)-dependent PFK1 and 1.7 kilounit/liter glycerol-3-phosphate dehydrogenase (Sigma). The reaction was started by the addition of 0.5 mmol/L pyrophosphate, and the change in absorbance followed at 339 nm. The F2,6P2 content was calculated and normalized relative to total cellular protein, as determined by the bicinchoninic acid (BCA) assay.
3
Enzymatic Cleavage of Deoxy-Ribose Substrates
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The cleavage of the natural DERA substrate, deoxyribose 5-phosphate (DRP), was measured using 2-deoxyribose 5-phosphate sodium salt (Sigma-Aldrich) as a substrate in a coupled enzyme system with triosephosphate isomerase (TPI) and glycerol 3-phosphate dehydrogenase (GPD) from rabbit muscle (Sigma-Aldrich) in ambient temperature. DERA activity on DRP liberates glyceraldehyde-3-phosphate, which is reduced to glycerol-3-phosphate by the supplementary enzymes TPI and GDH. The latter reaction consumes NADH, which can be detected by spectrophotometer. The reaction mixture contained 0.1 μM purified DERA wild-type or variant, 5 mM DRP, 3 units of TPI, 2 units of GPD and 0.3 mM NADH in 50 mM Tris-HCl, pH 7.5, supplemented with 5 mM MgCl2. The reaction was initiated by addition of DRP and followed by measuring the decrease of absorbance at 340 nm using a Varioskan microtiter plate reader (Thermo). DERA activity on non-phosphorylated substrate 2-deoxy-D-ribose (DR, Sigma-Aldrich) was assayed similarly in a coupled enzyme system with 4 units of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (Sigma-Aldrich) using 50 mM DR and 2 μM purified DERA wild-type enzyme or variant, and 0.3 mM NADH in 50 mM Tris-HCl, pH 7.5, supplemented with 5 mM MgCl2. Cleavage of DR by DERA liberates acetaldehyde, which is converted to ethanol by ADH in NADH consuming reaction.
4
Intracellular F2,6BP Quantification Protocol
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Intracellular F2,6BP concentration was determined as previously described.32 (link) Briefly, cells were centrifuged at 200 × g, resuspended in 20 volumes of 0.05 N NaOH and then 1 volume of 0.1 N NaOH to obtain a pH >11, vortexed for 10 s, incubated at 80 °C for 5 min and cooled in an ice bath. Cell extracts were neutralized to pH 7.2 with ice-cold acetic acid in the presence of 20 mM Hepes. Samples were incubated at 25 °C for 2 min in the following assay mixture: 50 mM Tris, 2 mM Mg+2, 1 mM F6P, 0.15 mM NAD, 10U/l PPi-dependent PFK-1, 0.45 kU/l aldolase, 5 kU/l triosephosphate isomerase and 1.7 kU/l glycerol-3-phosphate dehydrogenase (Sigma). In total, 0.5 mM pyrophosphate was added and the rate of change in absorbance (OD=339 nm) per min was followed for 5 min. F2,6BP was calculated based on a calibration curve produced by measuring 0.1 to 1 pmol of F2,6BP (Sigma) and normalized to total cellular protein.
5
Quantifying Tissue F2,6-BP Levels
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Tissue F2,6-BP concentration was determined as previously described [35 (link)]. Briefly, samples of LV were weighed and homogenized in NaOH (0.05 M). The resulting mixture was heated for 20 min at 80 °C. After cooling, the samples were neutralized with 1 M acetic acid in the presence of 20 mM Hepes and then centrifuged. Samples were incubated at 37 °C for 5 min in the following assay mixture: 50 mM Tris, 5 mM Mg2+, 1 mM fructose-6-phosphate (Sigma #F3627), 0.15 mM NADH (Sigma #N4505), excessive PPi-dependent PFK-1 (enriched from potato tubers), 0.2 U/mL aldolase (Sigma #A2714), 8 U/mL triosephosphate isomerase (Sigma #T2507), and 1 U/mL glycerol-3-phosphate dehydrogenase (Sigma #10127752001). After the 5 min pre-incubation time, 0.5 mM pyrophosphate was added to start the reaction, and the rate of change in OD340nm every 30 s was followed for 5 min in a Bio-Rad xMark microplate spectrophotometer (Bio-Rad, Hercules, CA, USA). Data are expressed as the fold change compared to the WT controls.
6
Quantifying Fructose-2,6-bisphosphate Levels
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Fructose-2,6-bisphosphate levels were determined based on the activation of pyrophosphate-dependent PFK1, as previously described42 (link). Briefly, cells were pelleted by low-speed centrifugation and resuspended in a solution containing 20 volumes of 50 mM NaOH and 1 volume of 100 mM NaOH (pH 11.0) and vortexed for 10 seconds. The solution was then heated at 80 °C for 5 minutes before being placed on ice, and the solution was neutralized to pH 7.2 with cold acetic acid in 20 mM HEPES buffer. Sample extracts were then incubated at 25 °C for 2 minutes in a solution containing 50 mM Tris, 2 mM Mg2+, 1 mM Fru-6P, 15 μM NAD, 10 units/liter PP-dependent PFK1 enzyme, 0.45 kilounits/liter aldolase, 5 kilounits/liter triose phosphate isomerase, and 1.7 kilounits/liter glycerol-3-phosphate dehydrogenase (Sigma). Then, 0.5 mM pyrophosphate was added, and the rate of change in absorbance (OD 339 nm) per minute was measured for 5 minutes. The F-2,6BP concentration was calculated based on a calibration curve ranging from 0.1 to 1.0 pmol of purified F-2,6BP (Sigma) and then normalized to total protein content.
7
Amino Acid Synthesis Protocols
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Chemicals used were: ammonium chloride, L-Arg hydrochloride, L-Ala, ethanol (99.5%), L-Gln, Gly, L-Orn hydrochloride, L-Ser, sodium L-Asp, sodium hydrogen L-Glu, sodium L-lactate (solution), sodium Pyr, Suc, and L-threonine from Wako Pure Chemical Industries, Ltd., Osaka, Japan, or Nacalai Tesque, Inc., Kyoto, Japan; L-Orn L-Asp (L-ornithine L-aspartate salt) from Tokyo Chemical Industry Co. Ltd., Tokyo; L-lysine hydrochloride and L-Pro from Sigma-Aldrich Inc., Japan, Osaka, Japan; MCT (78.9% powder) from Kissei Pharmaceutical Co. Ltd, Nagano, Japan. Glutamate dehydrogenase, glycerol-3-phosphate dehydrogenase and lactate dehydrogenase were purchased from Sigma-Aldrich Japan, Osaka, Japan.
8
Enzymatic Assay for Fructose-1,6-bisphosphate
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Enzymatic measurements of FBP were conducted according to the described procedure [2] . Briefly, the neutralized extract was added in the reaction buffer that contains 0.5 mM Na-ethylenediaminetetraacetic acid, 50 mM Tris-HCl (pH 7.5), and 0.2 mM NADH. Sequential addition of 0.2 U glycerol-3-phosphate dehydrogenase (Sigma, USA), 1 U triosephosphate isomerase (Sigma, USA), and 0.2 U aldolase (Sigma, USA) catalyzes the reactions. Absorbance was recorded at 340 nm at room temperature by using a spectrophotometer.
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