Cytoplasm samples outlined above were used for further ACC1 enzyme activity measurement. ACC1, rather than ACC2, was measured based on a discontinuous spectrophotometric method. Enzyme activity of ACC1 was determined as follows (Willis et al., 2008 ; Sumiya et al., 2015 (link)): 10 μl of ACC1-containing cytoplasm was added into pre-warmed (30°C) ACC reaction buffer [100 mM of potassium phosphate (Sigma-Aldrich, pH 8.0), 15 mM of KHCO3 (Sangon), 5 mM of MnCl2 (Sigma-Aldrich), 1 mg/mL of bovine serum albumin (BSA, Beyotime Biotechnology, China), 1 mM of acetyl-CoA (Sigma-Aldrich), 5 mM of ATP (Sigma-Aldrich), and 3 mg/L of biotin (Sigma-Aldrich)] to start the conversion of acetyl-CoA to malonyl-CoA in 30°C for 15 min. The untransformed acetyl-CoA in ACC reaction buffer was determined by incubating the cytoplasm-containing ACC reaction buffer with acetyl-CoA assay buffer [100 mM of potassium phosphate (pH 8.0), 0.1 mg/mL of dithionitrobenzoic acid (DTNB, Sigma-Aldrich), 20 mM of oxaloacetate (Sigma-Aldrich), and 1 mg/mL of BSA, 0.5 unit citrate synthase (Sigma-Aldrich)] at 30°C until the absorbance at 412 nm was not changed. The relative ACC1 activity normalized to overall cytoplasm protein was calculated by [OD412 (ACC reaction buffer)-OD412 (ACC reaction buffer with cytoplasm proteins)]/15 min/μg of cytoplasm protein.
Acetyl coa
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, China
Acetyl-CoA is a fundamental metabolic intermediate that plays a critical role in various cellular processes. It is the key entry point into the citric acid cycle, a central pathway in cellular respiration and energy production. Acetyl-CoA is involved in the synthesis of fatty acids, cholesterol, and other important biomolecules. It serves as a substrate for a wide range of enzymatic reactions, making it essential for maintaining cellular homeostasis and supporting diverse metabolic functions.
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Lab products found in correlation
Malonyl coa, by Merck Group (31 mentions)
Nadph, by Merck Group (19 mentions)
Butyryl coa, by Merck Group (9 mentions)
Adenosine triphosphate (atp), by Merck Group (8 mentions)
Oxaloacetic acid, by Merck Group (7 mentions)
Hexanoyl coa, by Merck Group (5 mentions)
Oleoyl coa, by Merck Group (5 mentions)
Propionyl coa, by Merck Group (5 mentions)
T4 dna ligase, by New England Biolabs (5 mentions)
Oxaloacetate, by Merck Group (5 mentions)
Acetoacetyl coa, by Merck Group (5 mentions)
Benzoyl coa, by Merck Group (4 mentions)
Octanoyl coa, by Merck Group (4 mentions)
Antarctic phosphatase, by New England Biolabs (4 mentions)
Fatty acid, by Merck Group (4 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Acetonitrile, by Thermo Fisher Scientific (4 mentions)
Methanol, by Thermo Fisher Scientific (4 mentions)
Palmitoyl coa, by Merck Group (3 mentions)
152 protocols using acetyl coa
1
Assaying Acetyl-CoA Carboxylase 1 Activity
2
Acetylation Modulates C. elegans Development
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Worms were injected at 6 h post-L4 stage, and the injected worms were fixed and immunostained at 18 hours post injection. For Trichostatin A (TSA, Sigma) treatment, a concentration of 10 μM TSA was used and 0.2% DMSO (v/v) was injected as control. For Acetyl-CoA (Sigma) injection, a concentration of 100 μM Acetyl-CoA was used, and H2O was injected as control.
3
Reconstitution of Lipid Biosynthesis Pathways
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1-palmitoyl-2-oleoyl-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphate (14:0 Lyso PA), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (16:0 Lyso PA), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate (18:0 Lyso PA), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (18:1 Lyso PA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) were purchased from Avanti Polar lipids. Fatty acid LC/MS mixture (#17942) was purchased from Funakoshi (Japan). 13C-Acetyl-CoA, 13C-Malonyl-CoA, Acetyl-CoA, Malonyl-CoA, NADH, NADPH, and sn-glycerol-3-phosphate were purchased from Sigma Aldrich. PUREfrex 2.0 and its Sol.I buffer, which was customized for the volume down, and DnaKJE mix were given or purchased from GeneFrontier (Japan).
4
Chemoenzymatic Synthesis of Acyl-Carrier Protein
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The restriction enzymes, T4 DNA ligase, oligonucleotide
primers,
and the competent E. coli BL21(DE3) cells were purchased
from Invitrogen. Pfu Turbo and Deep Vent DNA polymerases were purchased from Strategene. The cloning vectors
were from Novagen. DNA sequencing was performed by the DNA Sequencing
Facility of the University of New Mexico. Acetyl-CoA, benzoyl-CoA,
propanoyl-CoA, hexanoyl-CoA, lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA,
and oleoyl-CoA were purchased from Sigma. The thioester substrates
4-hydroxybenzyol-CoA, 3-hydroxybenzoyl-CoA, 1,4-dihydroxynapthoyl-CoA,
3-hydroxyphenylAcetyl-CoA, and coumaroyl-CoA were synthesized as previously
reported.18 (link),21 (link)E. coli strains JW1676
(ΔydiI::kanr) and BW25113 (wild-type)
of the Keio collection were obtained from Yale University.22 (link) The engineered E. coli strain
DK574, carrying the plasmid pJT93 expressing the E. coli AcpS transferase gene, under tac-promoter control,
was a kind gift from Dr. John Cronan of the University of Illinois.
The holoACP (UniProt accession code P0A6A8) purified
from this strain was converted to benzoyl-holoACP by using the chemical
procedure reported in ref (23 (link)). The molecular mass and purity of the isolated adduct were
verified by ES-MS analysis.
primers,
and the competent E. coli BL21(DE3) cells were purchased
from Invitrogen. Pfu Turbo and Deep Vent DNA polymerases were purchased from Strategene. The cloning vectors
were from Novagen. DNA sequencing was performed by the DNA Sequencing
Facility of the University of New Mexico. Acetyl-CoA, benzoyl-CoA,
propanoyl-CoA, hexanoyl-CoA, lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA,
and oleoyl-CoA were purchased from Sigma. The thioester substrates
4-hydroxybenzyol-CoA, 3-hydroxybenzoyl-CoA, 1,4-dihydroxynapthoyl-CoA,
3-hydroxyphenylAcetyl-CoA, and coumaroyl-CoA were synthesized as previously
reported.18 (link),21 (link)E. coli strains JW1676
(ΔydiI::kanr) and BW25113 (wild-type)
of the Keio collection were obtained from Yale University.22 (link) The engineered E. coli strain
DK574, carrying the plasmid pJT93 expressing the E. coli AcpS transferase gene, under tac-promoter control,
was a kind gift from Dr. John Cronan of the University of Illinois.
The holoACP (UniProt accession code P0A6A8) purified
from this strain was converted to benzoyl-holoACP by using the chemical
procedure reported in ref (23 (link)). The molecular mass and purity of the isolated adduct were
verified by ES-MS analysis.
5
Biochemical Assay Protocol Compendium
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Bio-Rad protein assay kit was purchased from Bio-Rad; sodium pyruvate, malic acid, succinic acid, ascorbic acid, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), palmitoyl-L-carnitine, rotenone, antimycin A, oligomycin, coenzyme A trilithium salt (CoA-SH), acetyl-CoA, oxaloacetic acid, thiamine pyrophosphate (TPP), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), 2,6-dichlorophenolindophenol (DCPIP), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (CCCP), phosphoenolpyruvate (PEP), ADP, ATP, NAD+, NADH, NADPH, KCN, pyruvate kinase, lactate dehydrogenase, cytochrome c, and decylubiquinone were from Sigma; KO was a generous gift of Aker BioMarine ASA (Oslo, Norway). Antibodies against AAC and UCP2 were from Santa Cruz Biotechnology (sc-11433 and sc-6526); antibodies against OXPHOS proteins were from Mitosciences (ab110413). Kits for the assay of triglycerides and total cholesterol were purchased from Futura System. Plasma insulin concentration was analyzed with a Mercodia Ultrasensitive Mouse Insulin kit. Luciferase ATP assay kit was from Sigma and Lipid Hydroperoxide (LPO) assay kit was from Merck. All other reagents were of analytical grade.
6
Kinetic Analysis of Acyl-CoA Acetyltransferase
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Varied concentrations of acetyl-CoA (Sigma Aldrich, Cat# A2181), methacrylyl-CoA and crotonyl-CoA were incubated with 0.1 µM HAT1 and 200 µM of H4 (1–20) peptide (sequence: Ac-SGRGKGGKGLGKGGAKRHRK). The enzymatic reactions were conducted at 30 °C for 15 min in KAT reaction buffer containing 50 mM HEPES-Na and 0.1 mM EDTA-Na, pH 8.0. The reactions were quenched by 50% isopropanol and incubated with the probe 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (abbr. CPM, Thermo Fisher, Cat# D346) in the darkness for 30 min at room temperature, producing the fluorescent CPM-SCoA complex. The fluorescence intensities were measured at an excitation of 392 nm and an emission of 482 nm with FlexStation®3 microplate reader to obtain reaction rate for each datapoint. Kinetic constants including binding affinity (Km) and catalytic rate (kcat) were determined by fitting the acyl-CoA concentration- rate curve to the Michaelis-Menten equation using GraphPad Prism.
7
PD-L1 Acetylation Assay Protocol
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Recombinant His-tagged PD-L1 was purified from bacteria. 0.5 μg of His-PD-L1 and/or 0.5 μg of active p300 recombinant protein (#81158, Active motif) were incubated in acetylation assay buffer (50 mM Tris pH 8.0, 0.1 mM EDTA, 50 ng/μl BSA) in the presence or absence of 20 μM Acetyl-CoA (#10101893001, Sigma) for 1 hour at 30°C. For mass-spectrometric analysis, peptide (AA 261–270) was synthesized and purchased from FUJIFILM Wako and 0.5 mg of the peptide was used for the assay.
8
Acetylation and Deacetylation of Microtubules
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To acetylate microtubules in vitro (13 (link)), 20 μM 30% rhodamine-labeled porcine microtubules were incubated with 3 μM αTAT (recombinantly expressed and purified murine αTAT, which is also known as MEC-17 (10 (link)), was a generous gift from Wilhelm Walter and Stefan Diez, B CUBE–Center for Molecular Bioengineering, TU Dresden, Dresden, Germany) and 120 μM acetyl-coenzyme A (acetyl-CoA, Sigma-Aldrich) in 60 mM Tris-HCl, pH 8.0 for the indicated times at 28°C.
To deacetylate microtubules in vitro, Chlamydomonas tubulin at 17 μM was incubated with 50 μg/mL human recombinant His-Tag SIRT2 (EMD Millipore, Darmstadt, Germany), 0.5 mM NAD+ (Sigma Aldrich) in 40 mM PIPES with 0.5 mM DTT, 0.8 mM EGTA, and 0.5 mM MgSO4 at pH 7.0 at room temperature for 2 h. This deacetylated tubulin was further polymerized into microtubules as described above.
The controls for both cases were treated in the same manner, just without addition of αTAT or SIRT2. The microtubules were used for gliding assays, SDS-PAGE, and Western blots.
To deacetylate microtubules in vitro, Chlamydomonas tubulin at 17 μM was incubated with 50 μg/mL human recombinant His-Tag SIRT2 (EMD Millipore, Darmstadt, Germany), 0.5 mM NAD+ (Sigma Aldrich) in 40 mM PIPES with 0.5 mM DTT, 0.8 mM EGTA, and 0.5 mM MgSO4 at pH 7.0 at room temperature for 2 h. This deacetylated tubulin was further polymerized into microtubules as described above.
The controls for both cases were treated in the same manner, just without addition of αTAT or SIRT2. The microtubules were used for gliding assays, SDS-PAGE, and Western blots.
9
Mitochondrial PDH Activity Assay
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The PDH activity was determined in isolated mitochondria from fresh cells (Mitochondria Isolation Kit, BioVision) by using a coupled enzyme reaction, which results in a colorimetric product proportional to the enzymatic activity (BioVision). Acetyl-CoA (Sigma) was added directly to the mitochondrial lysates (10 mM) before the assay.
10
Intranasal Delivery of Labeled Compounds
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