Actoacetanilide was the product of the Tokyo Chemical Industry (Tokyo, Japan). S-Ketamine was obtained from Cayman Chemical Company (Ann Arbor, MI, USA) as a certified reference material (CRM) solution in methanol (1 mg/mL). Before using this reagent, the methanol solvent was evaporated under a stream of argon gas, and the chemical was redissolved in 0.1 M Hepes buffer pH 7.4 containing 60 mM KCl. The concentration of ketamine in the stock solution was determined from its absorbance at 265 nm using the extinction coefficient of 0.562 mM−1cm−1. (S)-norketamine hydrochloride and midazolam were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, NADP, Glucose-6-phosphate, and midazolam were the products of MilliporeSigma (Burlington, MA, USA). All other reagents were of ACS grade and used without additional purification.
Glucose 6 phosphate dehydrogenase from leuconostoc mesenteroides
Manufactured by Merck Group
Sourced in United States
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is an enzyme that catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone. This reaction is the first step in the pentose phosphate pathway, which is important for the generation of NADPH and the production of pentose sugars.
Automatically generated - may contain errors
Lab products found in correlation
Glucose 6 phosphate (g6p), by Merck Group (3 mentions)
Midazolam, by Cayman Chemical (1 mentions)
Clotrimazole, by Tokyo Chemical Industry (1 mentions)
D glucose 6 phosphate sodium salt, by Merck Group (1 mentions)
Catalase from bovine liver, by Merck Group (1 mentions)
D glucose 6 phosphate, by Merck Group (1 mentions)
Methotrexate, by Merck Group (1 mentions)
6r 5 10 dideazatetrahydrofolate ddthf, by Merck Group (1 mentions)
Quikchange multi kit, by Agilent Technologies (1 mentions)
β nicotinamide adenine dinucleotide phosphate sodium salt hydrate, by Merck Group (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Phloretin, by Merck Group (1 mentions)
Compound c, by Merck Group (1 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
β nadp, by Oriental Yeast (1 mentions)
8 protocols using glucose 6 phosphate dehydrogenase from leuconostoc mesenteroides
1
Quantitative analysis of ketamine and its metabolites
2
Mass Spectrometry Proteomic Assay
3
Isotopic DHFR Preparation for NMR
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Reduced β-nicotinamide adenine
dinucleotide phosphate tetrasodium salt hydrate (NADPH),d -glucose-6-phosphate sodium salt, and glucose-6-phosphate dehydrogenase
from Leuconostoc mesenteroides were
purchased from Sigma-Aldrich. (6S)-5,6,7,8-Tetrahydrofolic
acid (THF) was obtained from Schircks Laboratories. Expression and
purification of L28F DHFR were performed as described previously.6 (link),26 (link) Isotopically labeled L28F DHFR for proton relaxation dispersion
measurements was expressed in M9 medium containing 0.5 g/L of 15NH4Cl, 0.5 g/L 15NH4(SO4)2, and 3 g/L [13C,1H]-glucose
in 99% D2O according to published protocols.27 (link),28 (link)
dinucleotide phosphate tetrasodium salt hydrate (NADPH),
from Leuconostoc mesenteroides were
purchased from Sigma-Aldrich. (6S)-5,6,7,8-Tetrahydrofolic
acid (THF) was obtained from Schircks Laboratories. Expression and
purification of L28F DHFR were performed as described previously.6 (link),26 (link) Isotopically labeled L28F DHFR for proton relaxation dispersion
measurements was expressed in M9 medium containing 0.5 g/L of 15NH4Cl, 0.5 g/L 15NH4(SO4)2, and 3 g/L [13C,1H]-glucose
in 99% D2O according to published protocols.27 (link),28 (link)
4
Conversion of Myristic Acid by P450 Enzymes
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Conversions of myristic acid were performed at 30°C in a total reaction volume of 300 μl. Samples contained 50 mM KPi, pH 7.5, 1 mM MgCl2, 2% (v/v) DMSO, 200 μM myristic acid, 4 mM d -glucose 6-phosphate, and 3 U glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich) for cofactor regeneration, 600 U catalase from bovine liver (Sigma-Aldrich) to decompose hydrogen peroxide that can be produced within the P450 catalytic cycle, 200 μM NADPH, 1 μM P450 (BMO–HFBI or BMO), and 1 or 5 μM reductase (BMR–HFBI or BMR). Additionally, a reaction was set up that contained 1 μM holoenzyme P450 BM3 instead of BMO and BMR.
Analysis of reaction products was done by gas chromatography coupled with mass spectrometry (GC/MS). Samples were acidified with HCl, extracted with diethyl ether, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane, and analyzed, as described previously (Girhard et al., 2013 (link)).
Analysis of reaction products was done by gas chromatography coupled with mass spectrometry (GC/MS). Samples were acidified with HCl, extracted with diethyl ether, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane, and analyzed, as described previously (Girhard et al., 2013 (link)).
5
Kinetic Analyses of DHFR Variants
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β-Nicotinamide adenine dinucleotide
phosphate reduced tetrasodium salt hydrate (NADPH), β-nicotinamide
adenine dinucleotide phosphate sodium salt hydrate (NADP+),d -glucose-6-phosphate solution, glucose-6-phosphate dehydrogenase
from Leuconostoc mesenteroides, methotrexate,
folic acid and (6R)-5,10-dideazatetrahydrofolate
(ddTHF, also known as lometrexol hydrate) were purchased from Sigma-Aldrich.
(6S)-5,6,7,8-tetrahydrofolic acid (THF) was obtained
from Schircks Laboratories. The L28F DHFR was generated by site-directed
mutagenesis using the QuikChange Multi kit (Agilent) as described
elsewhere.29 (link) Plasmid construction, protein
expression, and purification of WT and L28F DHFRs were performed as
described previously.29 (link),30 (link) All experiments, including kinetic
measurements, were performed in NMR buffer (70 mM KPi,
25 mM KCl, 1 mM DTT, pH 7.6) unless otherwise specified.
phosphate reduced tetrasodium salt hydrate (NADPH), β-nicotinamide
adenine dinucleotide phosphate sodium salt hydrate (NADP+),
from Leuconostoc mesenteroides, methotrexate,
folic acid and (6R)-5,10-dideazatetrahydrofolate
(ddTHF, also known as lometrexol hydrate) were purchased from Sigma-Aldrich.
(6S)-5,6,7,8-tetrahydrofolic acid (THF) was obtained
from Schircks Laboratories. The L28F DHFR was generated by site-directed
mutagenesis using the QuikChange Multi kit (Agilent) as described
elsewhere.29 (link) Plasmid construction, protein
expression, and purification of WT and L28F DHFRs were performed as
described previously.29 (link),30 (link) All experiments, including kinetic
measurements, were performed in NMR buffer (70 mM KPi,
25 mM KCl, 1 mM DTT, pH 7.6) unless otherwise specified.
6
Expression and Purification of Ferredoxin:NADP+ Oxidoreductase
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B. subtilis ferredoxin:NADP+ oxidoreductase was prepared as previously described, and its concentration was determined spectrophotometrically according to ε457 = 12.3 mM−1cm−1 [16 (link)]. NADP(H), 3-acetylpyridineadenine dinucleotide phosphate (APADP+), horse heart cytochrome c, superoxide dismutase, glucose oxidase, catalase, glucose 6-phosphate, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, 5-deaza-FMN, and other commercially available reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as received.
7
Recombinant Human P450 Enzyme Assay
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Phloretin, the oxidized form of β-nicotinamide adenine dinucleotide phosphate (NADP+), glucose-6-phosphate, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, acetonitrile, methanol, and ethyl acetate were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other chemicals used in this work were obtained with the highest grade commercially available, and they were used without further purification.
Recombinant human P450s were heterologously expressed in E. coli with a pCW vector containing human P450 cDNA (CYP3A4 or CYP2C19) and rat NADPH-P450 reductase (CPR) [34 (link),35 (link)]. pCW vectors expressing P450 and CPR were constructed in previous studies: CYP1A2, CYP3A4, CYP2C19, CYP1B1, CYP2E1, CYP2D6, and CYP2A6 [34 (link),35 (link)]. Membrane fractions expressing P450 and CPR were prepared, as described previously [34 (link),35 (link)], and were used for the catalytic activity assays.
HLMs (a human liver microsome pool) were purchased from ThermoFisher Scientific (Walthan, MA, USA).
Antibodies against CYP3A4 and 2C19 were prepared in the previous work [36 (link)].
Recombinant human P450s were heterologously expressed in E. coli with a pCW vector containing human P450 cDNA (CYP3A4 or CYP2C19) and rat NADPH-P450 reductase (CPR) [34 (link),35 (link)]. pCW vectors expressing P450 and CPR were constructed in previous studies: CYP1A2, CYP3A4, CYP2C19, CYP1B1, CYP2E1, CYP2D6, and CYP2A6 [34 (link),35 (link)]. Membrane fractions expressing P450 and CPR were prepared, as described previously [34 (link),35 (link)], and were used for the catalytic activity assays.
HLMs (a human liver microsome pool) were purchased from ThermoFisher Scientific (Walthan, MA, USA).
Antibodies against CYP3A4 and 2C19 were prepared in the previous work [36 (link)].
8
Fmoc Synthesis of WH and HW Peptides
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WH and His-Trp (HW) were synthesized using an Fmoc solid-phase synthesis method according to the manufacturer’s instructions (Kokusan Chemicals, Tokyo, Japan). AICAR and insulin were obtained from Wako Pure Chemical Industries (Osaka, Japan). LKB1 (siRNA ID: s163339) and PHT1 (siRNA ID: s140941) siRNA oligonucleotides were purchased from Life Technologies (Tokyo, Japan). Fluo-4AM was obtained from Dojindo (Kumamoto, Japan). Compound C, 2-deoxy-d -glucose (2DG), 2DG-6-phosphate sodium salt, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, and resazurin sodium salt were obtained from Sigma–Aldrich (St. Louis, MO, USA). β-NADP+ and diaphorase from Clostridium kluyveri were obtained from Oriental Yeast (Tokyo, Japan). The various antibodies used in this study were obtained from Cell Signaling Technology (Tokyo, Japan; phospho-AMPKα (Thr172, No. 2531), AMPKα (Nο. 2532), β-actin (No. 4967), phospho-Akt (Ser473, No. 4060), Akt (No. 4691), insulin receptor substrate-1 (IRS-1, No. 2382), LKB1 (No. 3047), phospho-CaMKII (Thr286, No. 3361), and CaMKII (No.4436)), Santa Cruz Biotechnology (Dallas, TX, USA; Glut1, No. SC-7938), Life Technologies (Tokyo, Japan; phospho-IRS-1 (Tyr612, 44816G)), or Abcam (Tokyo, Japan; Glut4, ab65267).
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