The spectrophotometric method described by Clinkenbeard et al (23 (link)) was used with modifications. Each protein sample (16.4-43.2 µg) was incubated in 900 µl enzyme assay buffer (100 mM Tris-HCl, 100 µM EDTA, 0.2% v/v Triton X-100, pH 8.2) containing 300 µM acetyl-CoA (Sigma-Aldrich; Merck KGaA) for 17 min at 30˚C to prevent inactivation of HMGCS2 by succinylation (24 (link),25 (link)). Next, 35 nmol acetoacetyl-CoA (Sigma-Aldrich; Merck KGaA) were added, where HMGCS2 activity was calculated from the rate of decrease of acetoacetyl-CoA measured by spectrophotometry at 300 nm, at which the absorbance of the enolate form of acetoacetyl-CoA was maximum (26 ). The molar extinction coefficient of acetoacetyl-CoA is 3.6x103 in this enzyme assay buffer (23 (link)). Activity measurements were performed in three independent experiments. Data are expressed as the mean ± SD.
Acetoacetyl coa
Manufactured by Merck Group
Sourced in United States
Acetoacetyl-CoA is a chemical compound that is a key intermediate in the metabolism of fatty acids and ketone bodies. It serves as a precursor for the synthesis of various biomolecules, such as cholesterol, steroid hormones, and other lipids.
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Lab products found in correlation
Acetyl coa, by Merck Group (4 mentions)
Malonyl coa, by Merck Group (3 mentions)
Acetoacetyl coa, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Isoleucine, by Merck Group (1 mentions)
Blasticidin s, by Funakoshi (1 mentions)
Hygromycin b, by Nacalai Tesque (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Carbenicillin, by Nacalai Tesque (1 mentions)
Cobas fara centrifugal analyzer, by Roche (1 mentions)
Bl21 de3, by Tiangen Biotech (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Oxaloacetic acid, by Merck Group (1 mentions)
Succinyl coa, by Merck Group (1 mentions)
Arachidonoyl coa, by Merck Group (1 mentions)
Palmitoyl coa, by Merck Group (1 mentions)
Fluorometric acetyltransferase activity assay kit, by Abcam (1 mentions)
Hitrap q strong anion exchange column, by Bio-Rad (1 mentions)
Oligonucleotide primers, by Takara Bio (1 mentions)
Ni agarose columns, by Thermo Fisher Scientific (1 mentions)
16 protocols using acetoacetyl coa
1
Spectrophotometric Assay for HMGCS2 Activity
2
Purification and Kinetic Characterization of Acetoacetyl-CoA Reductase
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Overexpression of phaB genes in E. coli BL21(DE3) harboring the expression plasmids with IPTG induction, and purification of the N-His6-tagged recombinant proteins using Ni-affinity chromatography were carried out as described in Supporting Information. NADPH-dependent acetoacetyl-CoA reductase activity was assayed in the mixture composed of 200 μM NADPH, 1 to 20 μM acetoacetyl-CoA (Sigma-Aldrich, St. Louis, MO, USA), and enzyme solution with appropriate dilution in 200 μL of 100 mM Tris–HCl buffer (pH 8.0). The consumption of NADPH accompanied by decrease in absorbance at 340 nm was monitored at 30 °C (ε340 = 6.22 × 103 M−1 cm−1).
3
Kinetic Analysis of MabA Enzyme
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NADPH and acetoacetyl-CoA were purchased from Sigma-Aldrich. The reductase activity was determined by monitoring the decrease in the optical density at 340 nm corresponding to the oxidation of NADPH to NADP + . Kinetic constants for NADPH, acetoacetyl-CoA and -keto-octanoyl-CoA were determined with a fixed concentration of one component (1 mM for NADPH and 2 mM for acetoacetyl-CoA) and a range of concentrations of the second substrate. Assays were performed in a volume of 20 ml to determine the kinetic constants for acetoacetyl-CoA and -keto-octanoyl-CoA or in 80 ml for NADPH. Reaction mixtures consisted of 50 mM potassium phosphate buffer pH 7.0, the concentrations of substrates stated above and between 0.25 and 0.1 mM MabA MSMEG . The enzymatic reactions were initiated by the addition of acetoacetyl-CoA after incubation for 5 min at 25 C. The oxidation of NADPH was followed by removing 1.5 ml aliquots of the reaction mixture for analysis in a NanoDrop 2000c spectrophotometer (path length 1 mm; Thermo Scientific) to determine the kinetic constants of the -ketoacyl-CoAs and by continuous reading in an 80 ml quartz cuvette to determine the K m for NADPH in a NanoDrop 2000c spectrophotometer (path length 10 mm; Thermo Scientific). Experiments were performed in triplicate. Data were fitted using nonlinear least-squares regression with GraphPad Prism.
4
Isoleucine Biosynthesis Pathway
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Isoleucine, CoA, acetoacetyl-CoA, acetyl-CoA, malonyl-CoA, ATP and TeA were purchased from Sigma-Aldrich (St Louis, MO, USA). Kanamycin (Km), carbenicillin and hygromycin B were purchased from Nacalai (Kyoto, Japan). Blasticidin S was purchased from Funakoshi (Tokyo, Japan). All other reagents were of analytical grade.
5
SCOT1 Enzymatic Activity Assay
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HL-1 cell pellets were prepared 48 h after transfection by washing with PBS, pelleting at 4 °C and 800 × g for 15 min and homogenizing for a succinyl-CoA:acetoacetate transferase (SCOT1) enzymatic activity assay. SCOT1 activity was measured as succinate-induced decrease in absorbance at 303 nm using a medium of the following composition: 100 mmol/l Tris-H2SO4 (pH = 8.05), 25 mmol/l MgSO4, 50 µmol/l acetoacetyl-CoA (Sigma-Aldrich) and 0.1% (w/v) Triton X-100. Reactions were started by addition of sodium succinate at a final concentration of 50 mmol/l and absorbance at 303 nm was subsequently followed in time on a COBAS-FARA-centrifugal analyzer (Hoffmann-LaRoche).
6
Enzymatic Synthesis of Acyl Diketide-NACs
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Coenzyme A (CoA), fatty
acyl-CoA, acetoacetyl-CoA, malonyl-CoA, and SNAC were obtained from
Sigma-Aldrich. Acyl diketide-NACs with various lengths were synthesized
according to the published methods.25 (link),26 (link) Oligonucleotides
were obtained from Sangon Biotech (Shanghai) Co., Ltd. The expression
vector pET-CsyB was stored at a −80 °C freezer in our
laboratory.
acyl-CoA, acetoacetyl-CoA, malonyl-CoA, and SNAC were obtained from
Sigma-Aldrich. Acyl diketide-NACs with various lengths were synthesized
according to the published methods.25 (link),26 (link) Oligonucleotides
were obtained from Sangon Biotech (Shanghai) Co., Ltd. The expression
vector pET-CsyB was stored at a −80 °C freezer in our
laboratory.
7
Site-Directed Mutagenesis Protocol
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Primers used in site-directed mutagenesis were synthesized by Invitrogen Inc. (Shanghai, China). Mutations were confirmed by DNA sequencing with an ABI Genetic Analyzer 3730 (Invitrogen Inc., Shanghai, China). Escherichia coli strains DH5α and BL21 (DE3) were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The AxyPrep Plasmid Miniprep Kit was from Axygen Biotech Ltd. (USA). The pEVOL-ONBYRS plasmid was a gift from Professor Peter. G. Schultz at Scripps Research Institute (La Jolla, CA USA). The UAAs 4-cyano-l -phenylalanine, 4-methoxy-l -phenylalanine, 4-phenyl-L-phenyalanine and O-tert-butyl-l -tyrosine were purchased from Adamas Reagent Co., Ltd. (Switzerland). Isopropyl-β-d -thiogalactopyranoside (IPTG), NADH, and acetoacetyl-CoA were purchased from Sigma Chemical Co. (St. Louis, USA).
8
Enzymatic HADH Activity Measurement
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HydroxyacylCoA dehydrogenase (HADH) activity: 2.5 ml of total cell extract were incubated at 25°C in 133 mM triethanolamine pH7.0, 10 mM aceto-acetyl-CoA, 3.4 mM NADH, 20 mM KCN and 100 mM EDTA (all from Sigma-Aldrich). The formation of NAD generated from NADH was evaluated by following the absorbance at 340 nm over 5 min as previously described [42] (link). HADH activity was normalized to protein quantity and expressed relatively to HADH activity in control non-infected SLC at 24 h that level was set to 1.
9
Enzymatic Assays for Citrate Synthase and β-Hydroxyacyl-CoA Dehydrogenase
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Citrate synthase (CiS) and β-hydroxyacyl-CoA dehydrogenase (β-HAD) activities were measured as previously described [16 (link)]. Frozen samples were homogenised in a buffer (pH 7.4) containing 175 mmol/l KCl and 2 mmol/l EDTA. For CiS activity, tissue homogenates were mixed with a working solution containing 450 μM acetyl-CoA (Sigma–Aldrich, #A2056) and 0.1 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Sigma–Aldrich, #D218200), and absorbance was measured at 405 nm as blank values. Oxaloacetic acid (Sigma–Aldrich, #O4126, final concentration 1 mM) was then added to the assay system to initiate the reaction. The enzyme activity was calculated based on the slope of the absorbance curve. For β-HAD activity, tissue homogenates were mixed with a working solution containing 0.2 mM NADH (Sigma–Aldrich, #N4505) and absorbance at 340 nm was measured as the blank. Acetoacetyl-CoA (Sigma–Aldrich, #A1625, final concentration 0.1 mM) was then added to the assay system to initiate the reaction. The enzyme activity was calculated based on the slope of the absorbance curve and the extinction coefficient of NADH (6.22 µmol−1.cm−1).
10
Enzyme-Mediated Glycosylation with Diverse Compound Libraries
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In this contribution, four compound libraries, namely sugar acceptor, sugar donor, acyl acceptor and acyl donor libraries, were provided for enzyme-mediated reactions. The compounds listed in Fig. 1 and Supplementary Information Fig. S4 include diverse structures like steroids (1 —24 ), flavonoids (25 —31 ), alkaloids (32 —38 ), triterpenoids (39 —42 ), phenolic acids (43 —47 ) and coumarins (48 —49 ) are used as the sugar acceptors for OsSGT1-catalyzed glycosylation reactions (Fig. 1 and Supplementary Information Fig. S4 ). The sugar donors consist of seven UDP-activated nucleotides, among which, UDP-D-glucose (UDP-Glc), UDP-D-galactose (UDP-Gal), UDP-D-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc) were obtained from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). UDP-D-xylose (UDP-Xyl), UDP-l-arabinose (UDP-Ara) and UDP-D-galacturonic acid (UDP-GalA) was synthesized by enzyme-mediated reactions in our laboratory27 (link), 28 , 29 (link). The acyl acceptor library is made up of 10 steroidal glucosides (1a —10a ) and 13 other glucosides (50 —62 ) listed in Supplementary Information Fig. S58 . The acyl donor library includes acetyl-CoA, succinyl-CoA, arachidonoyl-CoA, palmitoyl-CoA and acetoacetyl-CoA, all of which were purchased from Sigma-Aldrich Co., LLC. The other chemicals were either reagent or analytic grade when available.
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