β nadph

Manufactured by Merck Group

Sourced in United States, Spain

β-NADPH is a form of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), which is a key cofactor involved in various biochemical processes. It serves as a reducing agent in numerous enzymatic reactions and plays a crucial role in cellular metabolism and energy production.

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48 protocols using β nadph

Antioxidant and Cytotoxic Assays Protocol

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Eagle’s minimum essential medium (MEM), Ham’s F12, Trypsin-EDTA, and trypan blue were purchased from Gibco BRL (USA). Fetal bovine serum (FBS; PAA Laboratories, Australia), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1, 1-diphenyl, 2-picrylhydrazyl (DPPH), ascorbic acid, glutathione (GSH), glutathione reductase (GR) and β-NADPH were purchased from Sigma (USA), 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) was purchased from Invitrogen (USA). Protease and phosphatase inhibitor cocktail, rabbit monoclonal anti-caspase-3, rabbit polyclonal anti-phospho-p53, mouse monoclonal anti-β-actin antibodies, biotinylated protein ladder, HRP-conjugated secondary anti-rabbit, and anti-mouse antibodies were purchased from Cell Signaling Technology (USA). Rabbit polyclonal anti-Nrf-2 (H-300) antibody was purchased from Santa Cruz Biotechnology (USA). Enhanced chemiluminescence (ECL) detection system was obtained from Pierce (USA). Polyvinylidene difluoride (PVDF) membrane and H₂O₂ were purchased from MERCK Millipore (Germany).

Khin Aung Z.M., Jantaratnotai N., Piyachaturawat P, & Sanvarinda P. (2022). A pure compound from Curcuma comosa Roxb. protects neurons against hydrogen peroxide-induced neurotoxicity via the activation of Nrf-2. Heliyon, 8(11), e11228.

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Cloning and Purification of Recombinant Proteins

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All restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs (MA, USA). Plasmid pET-30a was obtained from Novagen (Germany). Escherichia coli DH10β and BL21 (DE3) were used as the host for plasmid cloning and protein expression, respectively. Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen (USA). DNA and protein markers were acquired from New England Biolabs, β- NADPH, β-sitosterol and stigmasterol were acquired from Sigma-Aldrich (USA). Other materials used in this study were of analytical grade and were commercially available.

Bansal R., Sen S.S., Muthuswami R, & Madhubala R. (2019). A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B. PLoS Neglected Tropical Diseases, 13(4), e0007260.

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Synthesis and Characterization of NQC Compound

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N′-(Pyridine-3-carbonyl)quinoxaline-2-carbohydrazide (NQC) was synthesised and purified to 99.2% purity in International Medical University (IMU). Structure of the quinoxaline-2-carbohydrazide derivative was confirmed by spectroscopic methods. All solvents used in this research were of high performance column chromatography (HPLC) grade. Chemical reagents including formic acid and dipotassium phosphate (K₂HPO₄) from Fisher Scientific, dimethyl sulfoxide (DMSO) and monopotassium phosphate (KH₂PO₄) from Merck, and acetonitrile (ACN) from Friedemann Schmidt were used. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), sulphanilamide, N-(1-naphthyl)ethylenediamine and biological grade DMSO were procured from Sigma-Aldrich Sdn. Bhd, Malaysia. β-NADPH was procured from Sigma Aldrich, USA. RAW 264.7 cells and LPS (Escherichia coli O111:b4) were procured from American Type Culture Collection, Manassas, USA. HLM, RLM and MLM (20 mg/mL, Catalog #HMMCPL, Gibco) was procured from Life Technologies, Singapore.
Synthesis of N′-(pyridine-3-carbonyl)quinoxaline-2-carbohydrazide is as shown in Fig. 4.Fig. 4

Synthetic route of N′-(pyridine-3-carbonyl) quinoxaline-2-carbohydrazide (NQC)

Kandasamy M., Mak K.K., Devadoss T., Thanikachalam P.V., Sakirolla R., Choudhury H, & Pichika M.R. (2019). Construction of a novel quinoxaline as a new class of Nrf2 activator. BMC Chemistry, 13(1), 117.

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Oxidative Stress Enzyme Assay

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Benznidazole (BZL), dithionitrobenzoic acid, oxidized glutathione (GSSH), reduced glutathione (GSH), glutathione, nitroblue tetrazolium, MK571, β-NADPH, riboflavin, rifampicin (RIF), sulfosalicylic acid, tert-butyl hydroperoxide (tBOOH) and 2-vinylpyridine were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMSO and hydrogen peroxide were purchased from Merck (Darmstadt, HE, Germany). All other chemicals were of analytical grade purity.

Rigalli J.P., Perdomo V.G., Ciriaci N., Francés D.E., Ronco M.T., Bataille A.M., Ghanem C.I., Ruiz M.L., Manautou J.E, & Catania V.A. (2016). The antitripanocide Benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2). Toxicology and applied pharmacology, 304, 90-98.

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Antioxidant Assay with HOHA-Lactone

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Dulbecco’s modified Eagle’s medium (DMEM)/F12, Dulbecco’s phosphate buffered saline (DPBS), Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) were purchased from Fisher Scientific (Pittsburgh, PA). A human VEGF-A ELISA kit was purchased from Piercenet (ThermoFisher Scientific, Rockford, IL). Retinal pigmented epithelial cell basal medium (RtEBM), an optimized mixture of growth factors and supplements for primary hRPE cells (SingleQuots^™ Kit) was obtained from Lonza (Allendale, NJ). Goat anti rabbit FITC antibody and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Invitrogen (Carlsbad, CA). All other chemicals and reagents including L-glutathione (reduced), sodium borohydride, glutathione reductase (250 units ml⁻¹), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and β-NADPH, etc. were purchased from Sigma–Aldrich (St. Louis, MO). The lactone of 4-hydroxy-7-oxohept-5-enoic acid (HOHA-lactone) was synthesized as described previously.^{30 (link)} The purity of HOHA-lactone was 98.6% (¹H NMR).

Guo J., Linetsky M., Yu A.O., Zhang L., Howell S.J., Folkwein H.J., Wang H, & Salomon R.G. (2016). 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone Induces Angiogenesis through Several Different Molecular Pathways. Chemical research in toxicology, 29(12), 2125-2135.

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Visualizing NOS-Containing Neurons in GI Tract

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NADPH-d activity was detected histochemically to show the neurons containing NOS in the GI tract. Whole-mount preparations were washed in PBS, and then incubated in phosphate buffer (0.1 M, pH 7.4) containing 0.3% Triton X-100, 0.5 mg/mL nitroblue tetrazolium (Cat.No.N6876, Sigma-Aldrich, USA) and 1.0 mg/mL β-NADPH (Cat.No.N7505, Sigma-Aldrich, USA) for 1 h at 37 °C. The reaction was halted by placing the sample in PBS.

Sun T., Li D., Hu S., Huang L., Sun H., Yang S., Wu B., Ji F, & Zhou D. (2018). Aging-dependent decrease in the numbers of enteric neurons, interstitial cells of Cajal and expression of connexin43 in various regions of gastrointestinal tract. Aging (Albany NY), 10(12), 3851-3865.

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Preparation of Chemical Reagents

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Tunicamycin, ACh, SNP, phenylephrine, Tween-20, bis-N-methylacridinium nitrate, DETCA, DPI, β-NADPH, Tris-base were purchased from Sigma-Aldrich (St. Louis, MO, USA). DiOHF was purchased from Indofine and dissolved in DMSO. TUDCA was purchased from Calbiochem. Bovine serum albumin (BSA) was purchased from Santa Cruz (Dallas, Texas, USA). Kreb’s salts were purchased from BDH Limited and BDH Laboratory Supplies (Poole, UK).

Lau Y.S., Mustafa M.R., Choy K.W., Chan S.M., Potocnik S., Herbert T.P, & Woodman O.L. (2018). 3′,4′-dihydroxyflavonol ameliorates endoplasmic reticulum stress-induced apoptosis and endothelial dysfunction in mice. Scientific Reports, 8, 1818.

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Glutathione Reductase Activity in Immune Cells

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Glutathione reductase (GR) activity was assessed in peritoneal, spleen and thymus leukocytes following a method previously described [84 (link)] with some modifications [85 (link)]. Aliquots of leukocytes adjusted to 1 × 10⁶ cells/mL in Hank´s solution were centrifuged at 1000 g for 10 min at 4 °C and the supernatants were removed. Thereafter, leukocyte pellets were resuspended in 200 μL of 50 mM phosphate buffer 7.4 pH (Sigma-Aldrich) plus 6.3 mM ethylene-diaminetetracetic acid (EDTA, Sigma-Aldrich), previously degassed. Then, cells were sonicated and centrifuged at 3200 g for 20 min at 4 °C, and aliquots of the supernatant were used to evaluate GR. This method is based on the oxidation of β-NADPH (β-nicotinamide adenine dinucleotide 2-phosphate reduced) (6 mM, Sigma-Aldrich) due to the reduction of GSSG (80 mM, Sigma-Aldrich) by GR. The reaction was followed by spectrophotometry at 340 nm for 240 s. The results were expressed as milliunits (mU) of enzymatic activity per 10⁶ leukocytes.

Garrido A., Cruces J., Ceprián N., Vara E, & de la Fuente M. (2019). Oxidative-Inflammatory Stress in Immune Cells from Adult Mice with Premature Aging. International Journal of Molecular Sciences, 20(3), 769.

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Characterization of Cytochrome P450 Enzymes

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7-Hydroxy-4-(trifluoromethyl)coumarin (7-HFC), and 7-EFC were purchased from Life Technologies (Carlsbad, CA). β-NADPH, RNase A, DNase I, resorufin, and 7-BR were purchased from Sigma-Aldrich (St. Louis, MO). Ni²⁺-NTA affinity resin was purchased from Qiagen (Valencia, CA), and Macro-Prep CM cation exchange resin was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). The QuikChange XL site-directed mutagenesis kit and TOPP3 cells were obtained from Agilent Technologies (Santa Clara, CA). Recombinant NADPH:cytochrome P450 reductase (POR) [29 (link)] and cytochrome b₅ (b₅) from rat liver [30 (link), 31 (link)] were prepared as described previously. All other chemicals and supplies used were from standard sources. All protein model figures were created using MacPyMOL [32 ]. Channel/cavity analysis was performed using the Graphical User Interface for the Mole 2.0 program on Windows [33 (link)].

Jang H.H., Liu J., Lee G.Y., Halpert J.R, & Wilderman P.R. (2015). Functional Importance of a Peripheral Pocket in Mammalian Cytochrome P450 2B Enzymes. Archives of biochemistry and biophysics, 584, 61-69.

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Biochemical Reagents for Assays

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Tris, EDTA, HEPES, MOPS, NaCl, palmitoylCoA, rotenone, sucrose, 3-acetylpyridine adenine dinucleotide (APAD), and β-NADPH were all purchased from Sigma (St. Louis, MO). All reagents were of analytical grade or higher.

Fujisawa Y., Napoli E., Wong S., Song G., Yamaguchi R., Matsui T., Nagasaki K., Ogata T, & Giulivi C. (2014). Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency. BBA Clinical, 3, 70-78.

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