Triosephosphate isomerase

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Triosephosphate isomerase is a laboratory enzyme that catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, which are important intermediates in the glycolysis pathway. This enzyme plays a crucial role in the breakdown of glucose to generate energy for cellular processes.

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α-glycerophosphate dehydrogenase-triosephosphate isomerase (2 citations)

15 protocols using triosephosphate isomerase

Kinetic Characterization of PFK and GK

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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.

Gonzalez-Ordenes F., Cea P.A., Fuentes-Ugarte N., Muñoz S.M., Zamora R.A., Leonardo D., Garratt R.C., Castro-Fernandez V, & Guixé V. (2018). ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy. Frontiers in Microbiology, 9, 1305.

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Quantifying F2,6-BP in Tissue Samples

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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 OD_{340 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.

He X., Zeng H., Cantrell A.C., Williams Q.A, & Chen J.X. (2022). Knockout of TIGAR Enhances Myocardial Phosphofructokinase Activity and Preserves Diastolic Function in Heart Failure. Journal of cellular physiology, 237(8), 3317-3327.

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Enzymatic Assay for Phosphofructokinase-1

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The reaction was performed by using either cell lysate (30 μg) or recombinant purified PFKP (0.1 μg) in 1 ml of reaction buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM ATP, 0.2 mM NADH, 5 mM Na₂HPO₄, 0.1 mM AMP, 1 mM NH₄Cl, 5 mM fructose 6-phosphate (Sigma), 5 U of triose phosphate isomerase (Sigma), 2 U of aldolase (Sigma), and 1 U α-glycerophosphate dehydrogenase (Sigma). Absorbance was recorded at 339 nm at room temperature in a 96-well plate every 15 s for 10 min. In some experiments, PFK1 activity was determined by using a PFK activity colorimetric assay Kit (BioVision, Milpitas, CA).

Lee J.H., Liu R., Li J., Wang Y., Tan L., Li X.J., Qian X., Zhang C., Xia Y., Xu D., Guo W., Ding Z., Du L., Zheng Y., Chen Q., Lorenzi P.L., Mills G.B., Jiang T, & Lu Z. (2018). EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation. Molecular cell, 70(2), 197-210.e7.

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Enzymatic Activity Assay for UGT1a1 and SULT1a1

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UGT1a1 and SULT1a1 Supersomes were purchased from Corning (Netherlands). Sulfuric acid (96%), hydrogen peroxide (30%), sodium phosphate dibasic heptahydrate (98%), sodium phosphate monobasic monohydrate (98%), ethanol (95%), sodium hydroxide (98%), glutaraldehyde (25%), (3-aminopropyl)trimethoxysilane (97%), UDP-glucuronic acid (UDP-GA) (98%), 3′-phosphoadenosine-5′-phosphosulfate (PAPS, 60%), resorufin (95%), resorufin β-d-glucuronide (90%), p-nitrophenol (99%) and p-nitrophenyl sulfate (98%), LC-MS grade methanol, LC-MS grade water and triosephosphate isomerase, trifluoroacetic acid (TFA, 99%) were all obtained from Sigma-Aldrich (UK).

Doyle B., Madden L.A., Pamme N, & Jones H.S. (2023). Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites. RSC Advances, 13(40), 27696-27704.

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Phosphofructokinase Co-Substrate Specificity Assay

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To measure the co-substrate specificity of the phosphofructokinase, we used an assay according to Peng & Mansour [34 (link)] with modifications. The enzyme activity was determined in a coupled enzyme assay, with the standard assay master mix containing 50 mM Tris buffer (pH 8.5) with 1 mM EDTA and 1 mM DTT, 5 mM MgCl₂, 0.2 mg ml^-1 NADH, 1.5 U aldolase, 15 U triosephosphate isomerase, 5.1 U glycerolphosphat dehydrogenase (all from Sigma Aldrich, Germany), 1 mM Fru-6-P, 1 mM ATP and 1 mM PPi. The assays were run with the following controls: without Fru-6-P and without co-substrate ATP or PPi, respectively.

Kindzierski V., Raschke S., Knabe N., Siedler F., Scheffer B., Pflüger-Grau K., Pfeiffer F., Oesterhelt D., Marin-Sanguino A, & Kunte H.J. (2017). Osmoregulation in the Halophilic Bacterium Halomonas elongata: A Case Study for Integrative Systems Biology. PLoS ONE, 12(1), e0168818.

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Structural analysis of diverse proteins

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Triose phosphate isomerase (rabbit, TPI), avidin (chicken egg white, AVD), alcohol dehydrogenase (yeast, ADH), pyruvate kinase (rabbit muscle, PK), aldolase (rabbit muscle, ALD), transthyretin (human, TTR), Concanavalin A (jack bean, ConA), glutamate dehydrogenase (bovine liver, GDH) were purchased from Sigma Aldrich. A subset of these (TPI, AVD, ALD, TTR) were chosen for detailed IM-MS and CIU analysis, as they represent a broad cross-section of protein structures and stabilities. GDH was subjected to CXL treatment, but was not observed to undergo sufficient gas-phase unfolding following cross-linking, and thus not included in our CIU/CID analysis here. All other proteins listed were used for CCS calibration. Micro bio-spin columns with bio-gel P6 or P30 in a sodium chloride/citrate (SSC) buffer were purchased from Biorad.

Samulak B.M., Niu S., Andrews P.C, & Ruotolo B.T. (2016). Ion Mobility-Mass Spectrometry Analysis of Crosslinked Intact Multiprotein Complexes: Enhanced Gas-phase Stabilities and Altered Dissociation Pathways. Analytical chemistry, 88(10), 5290-5298.

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Enzymatic Assay for Metabolic Pathways

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Phenylmethylsulfonyl fluoride (PMSF), KH₂PO₄, NaCl, ethylenediamine tetraacetic acid (EDTA), ATP, ADP, MgCl_2, β-hydroxybutyrate, phosphoenolpyruvate, sodium L-lactate, sodium pyruvate, imidazole, hydrazine, aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, fructose 6-phosphate, glucose 6-phosphate, glucose, G6PDH, КСl, mannitol, bovine serum albumin, glutamate, malate, rotenone, succinate, antimycin A, ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), and NaN₃ were purchased from Sigma-Aldrich (St. Louis, MO, USA); Tris-HCl, NADP, NADH, NAD⁺, glycine, lactate dehydrogenase were from Carl Roth (Karlsruhe, Germany); diagnostic kits for determination of glucose and triacylglycerides were from Felicit Diagnostics Ltd. (Dnipro, Ukraine). All other reagents were obtained from local suppliers (Ukraine) and were of analytical grade.

Sorochynska O.M., Bayliak M.M., Gospodaryov D.V., Vasylyk Y.V., Kuzniak O.V., Pankiv T.M., Garaschuk O., Storey K.B, & Lushchak V.I. (2019). Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice. Frontiers in Physiology, 10, 1432.

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Enzymatic Assays for Glycolysis

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Clotrimazole, NAD⁺, NADH, NADP⁺, ATP, ADP, fructose 6-phosphate, lactate, glucose 6-phosphate, phosphoenolpyruvate, hexokinase, lactate dehydrogenase, aldolase, glucose 6-phosphate dehydrogenase, triosephosphate isomerase, and α-glycerophosphate were purchased from Sigma Chemical, St. Louis, MO, USA. Other reagents were of the highest purity available.

Marcondes M.C., Fernandes A.C., Itabaiana I J.r., de Souza R.O., Sola-Penna M, & Zancan P. (2015). Nanomicellar Formulation of Clotrimazole Improves Its Antitumor Action toward Human Breast Cancer Cells. PLoS ONE, 10(6), e0130555.

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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.

Yalcin A., Clem B.F., Imbert-Fernandez Y., Ozcan S.C., Peker S., O'Neal J., Klarer A.C., Clem A.L., Telang S, & Chesney J. (2014). 6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27. Cell Death & Disease, 5(7), e1337-.

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Enzymatic Assay of Fructose-6-Phosphate

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d-fructose-6-phosphate (Sigma-Aldrich), fructose-6-phosphate kinase pyrophosphate-dependent (0.1 U mL^–1, Sigma-Aldrich), aldolase (1 U mL^–1, Sigma-Aldrich), triosephosphate isomerase (5 U mL^–1, Sigma-Aldrich), glycerophosphate dehydrogenase (5 U mL^–1, Sigma-Aldrich), and NADH (Sigma-Aldrich).

Schoppet M., Peschke M., Kirchberg A., Wiebach V., Süssmuth R.D., Stegmann E, & Cryle M.J. (2018). The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03530j. Chemical Science, 10(1), 118-133.

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