Hexokinase

Manufactured by Merck Group

Sourced in United States, Germany

Hexokinase is an enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate, a key step in glycolysis. It plays a crucial role in the metabolism of carbohydrates.

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Hexokinase method (3 citations) Glucose (hexokinase) assay kit (8 citations) Hexokinase-dependent assay kit (2 citations) Hexokinase activity assay (2 citations) Hexokinase and glucose 6-phosphate dehydrogenase (2 citations) Hexokinase (from Saccharomyces cerevisiae Type F-300; (2 citations) Hexokinase activity kit (5 citations) Hexokinase Colorimetric Assay Kit (16 citations) Glucose hexokinase assay (2 citations) Hexokinase assay kit (3 citations)

56 protocols using hexokinase

Purifying and Activating Nucleotides

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ATP (A2383), AMP-PNP (A2647), ADP (A2754) were purchased from Sigma-Aldrich. The ATP regenerating system [200 μg/ml pyruvate kinase from rabbit muscle (Roche 10128155001), 2.5 mM Phosho(enol) pyruvic acid (Sigma P7252)] was mixed with ATP before use of ATP. The ADP (Sigma A2754) solution was treated with hexokinase (Sigma H4502) and D-glucose (1st BASE) to remove the contaminating ATP as described previously (34 (link)). The ADP solution (80 mM) was incubated with 200 mM D-glucose, 2 mM MgCl₂, 0.04 U/μl hexokinase (Sigma H4502-500UN) in room temperature for 2 h. After incubation, the enzyme was eliminated from these solution using a Amicon Ultra-0.5 column. The ADP·AlF was prepared by incubating 10 mM ATP-depleted ADP, 50 mM NaF (Sigma 67414) and 10 mM AlCl₃ (Sigma 563919) as described previously (35 (link)).

You H., Lattmann S., Rhodes D, & Yan J. (2016). RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis. Nucleic Acids Research, 45(1), 206-214.

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Mitochondrial function evaluation protocol

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ADP, Alamethicin, 3-amino-1,2,4-triazole, P1,P5-di(adenosine-5′)pentaphosphate (Ap5A), Amplex Red, ATP, carbonyl cyanide m-chlorophenylhydrazone (CCCP), cyclosporine A (CsA), EGTA, fatty acid-free BSA, glucose, glucose-6-phosphate dehydrogenase, glutamate, hexokinase, horseradish peroxidase, malate, mannitol, MgCl₂, NaCl, NADP, (NH₄)₂SO₄, oligomycin, Phenol Red, phosphoenolpyruvate, pyruvate kinase, rotenone, succinate, sucrose, tert-butyl hydroperoxide and Tris were from Sigma-Aldrich (Saint Louis, MO, USA); Coomassie G-250 was from MP Biomedicals (Santa Ana, CA, USA); CaCl₂, KCl, K₂HPO₄, KH₂PO₄ and Safranine O were from Merck (Darmstadt, Germany); Dihydroethidium, Mitotracker Green FM, Propidium Iodide and Sytox Green were from Life Technologies (Carlsbad, CA, USA). Other reagents of the highest purity available were from domestic suppliers. MitoQ, SkQ1 and SkQ3 were kindly provided by Dr. D.S. Esipov from the A.N. Belozersky Research Institute of Physico-Chemical Biology, MSU, Moscow, Russia.

Rogov A.G., Goleva T.N., Aliverdieva D.A, & Zvyagilskaya R.A. (2024). SkQ3 Exhibits the Most Pronounced Antioxidant Effect on Isolated Rat Liver Mitochondria and Yeast Cells. International Journal of Molecular Sciences, 25(2), 1107.

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ATP Depletion Protocol for Translation

Cited 1 time

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ATP depletion was performed as previously described [65 (link)]. Briefly, the translation products were incubated with 25 mM glucose and 0.5 units/μL hexokinase (Sigma) for 20 min at room temperature to deplete ATP in the reaction mix.

Jiang Z., Zhang K., Li Z., Li Z., Yang M., Jin X., Cao Q., Wang X., Yue N., Li D, & Zhang Y. (2020). The Barley stripe mosaic virus γb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes. PLoS Pathogens, 16(7), e1008709.

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Nano-FET Biosensor for Enzyme Assay

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Nano-FETs were immersed into a solution of 25% glutaraldehyde in water (Sigma-Aldrich) for at least 30 min while continuously sweeping the gate voltage and monitoring drain–source current. Afterward, also monitoring transistor performance, the probe was dipped into a solution of 500 U/mL hexokinase and glucose-6-phosphate dehydrogenase from S. cerevisiae (Sigma-Aldrich) for at least 30 min.

Zhang Y., Clausmeyer J., Babakinejad B., Córdoba A.L., Ali T., Shevchuk A., Takahashi Y., Novak P., Edwards C., Lab M., Gopal S., Chiappini C., Anand U., Magnani L., Coombes R.C., Gorelik J., Matsue T., Schuhmann W., Klenerman D., Sviderskaya E.V, & Korchev Y. (2016). Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis. ACS nano, 10(3), 3214-3221.

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Actin Polymerization Protocols

Cited 2 times

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Rabbit muscle actin, labeled actins, profilin, mouse cofilin-1, capping protein, and biotin-gelsolin were prepared as described in ref. ^{16 (link)}. ADP-actin was prepared as described in ref. ^{34 (link)}. Briefly, 30 µM ATP-G-actin solution containing 0.3 mM glucose and 1 unit/ml of hexokinase (Sigma) was dialyzed against nucleotide exchange buffer (5 mM Tris-HCl, 0.1 mM MgCl₂, 0.05 mM EGTA, 0.2 mM ADP, 1 mM DTT, pH 8.0) for 4 h at 4 °C. All experiments were performed using rabbit muscle α-actin.

Kotila T., Wioland H., Enkavi G., Kogan K., Vattulainen I., Jégou A., Romet-Lemonne G, & Lappalainen P. (2019). Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin. Nature Communications, 10, 5320.

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ADP-G-actin Fluorescence Assay

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To prepare ADP-G-actin, monomeric ATP-bound 2 mM rabbit muscle actin was treated overnight at 4°C with analytical grade anion exchange resin (BioRad), hexokinase (Sigma-Aldrich), and excess ADP. To initiate a reaction, 2 μM ADP-bound actin monomers and the indicated concentration of Cof2 or Cof2 ^V7M in CDT buffer (0.2 mM CaCl2, 0.2 mM DTT, 10 mM Tris pH 8.0), or buffer alone, were mixed and added to 50 μM ε-ATP. A fluorescence spectrophotometer (Photon Technology International) was used to monitor the reaction at 350-nm excitation and 410-nm emission at 25°C for 200 s.

Balakrishnan M., Yu S.F., Chin S.M., Soffar D.B., Windner S.E., Goode B.L, & Baylies M.K. (2020). Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth. Cell reports, 32(3), 107893.

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Glycogen Quantification in Soleus Muscle

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Frozen soleus muscle samples (5 mg) were homogenized (1/50) in 2 N NaOH for 2 hr at 37°C and 1 hr at 4°C and 0.2 volumes 7.5 M HCl added. Samples (50 µl) were subjected to glycogen hydrolysis by amyloglucosidase (10 mg/ml) (A7420, Sigma‐Aldrich) in acetate buffer (0.3 M) for 2 hr at 37°C. Released glucose was quantified by the spectrophotometric measurement of NADH production (λ = 340 nm) in the presence of hexokinase (H4502 Sigma) and glucose‐6‐phosphate dehydrogenase (G6378 Sigma), according to the method of Bergmeyer (Bergmeyer & Grassl, 1983 ; Passonneau & Lauderdale, 1974 (link)) and as previously described in (Banzet et al., 2009 (link)). Glycosyl units were quantified by comparison to a standard curve of known glycogen concentration.

Tardo‐Dino P., Taverny C., Siracusa J., Bourdon S., Baugé S., Koulmann N, & Malgoyre A. (2021). Effect of heat acclimation on metabolic adaptations induced by endurance training in soleus rat muscle. Physiological Reports, 9(16), e14686.

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Depletion of Residual ATP from Reagents

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Proteasomes were purified in the presence of ATP to preserve their integrity, and ubiquitinated conjugates were generated in the presence of ATP regeneration system. In addition, residual ATP contamination is often found in commercial ADP (EMD Millipore). Depletion of the ATP from these reagents is required to uncouple the effect of Ubp6 deubiquitination to proteasome degradation. To generate “ATP-free” ADP, powder was first dissolved in reaction buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl₂) and adjusted to pH 7.5 using 10 M KOH. The reconstituted ADP was then treated with 20 mM glucose and 0.2 U/μL hexokinase (Sigma) for 1 h at 30 °C. The final ADP concentration in solution was ~0.25 M. To generate ADP-protreasome, proteasomes (final ~0.5 μM) were hexokinase-treated in the same way. For ADP-HA-Ub_n-NCB1, ubiquitinated conjugates (final ~2.5 μM) were treated similarly but using 40 mM glucose and 0.4 U/μL hexokinase. 10% glycerol (v/v) was added for storage at −80 °C.

Hung K.Y., Klumpe S., Eisele M.R., Elsasser S., Tian G., Sun S., Moroco J.A., Cheng T.C., Joshi T., Seibel T., Van Dalen D., Feng X.H., Lu Y., Ovaa H., Engen J.R., Lee B.H., Rudack T., Sakata E, & Finley D. (2022). Allosteric control of Ubp6 and the proteasome via a bidirectional switch. Nature Communications, 13, 838.

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Metabolite Profiling of Drosophila Larvae

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Larval metabolic studies shown in Figure 1D were conducted using 25 staged late second instar larvae from at least three independent collections. Metabolite levels were normalized to protein levels and combined to determine average concentration and standard error of the mean, relative to the metabolite levels found in w¹¹¹⁸ controls. Triglyceride (TAG) measurements were performed using a coupled colorimetric assay (Sigma T2449) as described (Palanker et al., 2009 (link); Tennessen et al., 2014 (link)). Glycogen and glucose concentrations were determined using the Hexokinase (HK) and/or Glucose Oxidase (GO) assay kits (Sigma GAHK20, GAGO20) as described (Tennessen et al., 2014 (link)). Total protein levels were determined in parallel by Bradford assay. For adults, samples of five flies per replicate were collected at one week of age and homogenized in 100 µl of 1× PBS. Each assay was repeated at least three times for a combined total of 11–24 replicates per genotype, per sex. Metabolite levels were normalized to protein levels and combined to determine average concentration and standard error of the mean, relative to the metabolite levels found in btl-GAL4; UAS-DHR78 controls. Both control and tracheal-rescued mutant females were maintained with w¹¹¹⁸ males to ensure that mating took place.

Marxreiter S, & Thummel C.S. (2017). Adult functions for the Drosophila DHR78 nuclear receptor. Developmental dynamics : an official publication of the American Association of Anatomists, 247(2), 315-322.

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Phosphate Utilization Assay Protocol

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Phosphate utilization was assayed following a method published previously62 . In a total volume of 250 μl, an aliquot of 25 μl mitochondrial suspension was diluted into a medium containing 125 mM KCl, 75 mM sucrose, 0.1 mM EGTA, 1 mM MgCl₂, 10 mM HEPES, 2 mM phosphate, 0.3% BSA, 0.5 mM ADP (Sigma-Aldrich), 5 mM pyruvate, 10 mM succinate and 10 mM glucose, followed by immediate addition of 5 units of hexokinase (Sigma-Aldrich) and incubation at 37 °C for 30 min. The reaction was terminated by addition of 5% ice cold trichloroacetic acid (TCA) and the amount of inorganic phosphate was measured spectrophotometrically. A 0 min sample was also assayed for inorganic phosphate content where hexokinase addition was immediately followed by treatment with 5% ice cold TCA. Glucose and hexokinase in the reaction mixture acted as a trap for ATP to maintain the level of ADP in the system and also to prevent the release of free inorganic phosphate from ATP by the action of various phosphatases.

Maiti A.K., Sharba S., Navabi N., Forsman H., Fernandez H.R, & Lindén S.K. (2015). IL-4 Protects the Mitochondria Against TNFα and IFNγ Induced Insult During Clearance of Infection with Citrobacter rodentium and Escherichia coli. Scientific Reports, 5, 15434.

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