Coenzyme a trilithium salt

Recombinant
ACAT1 (200 ng/mL in
PBS) or vehicle was incubated with 2 mM MGO or vehicle for 2 h at
37 °C. 10 mM aqueous stocks of coenzyme A trilithium salt (CoA,
Sigma Aldrich) and acetoacetyl coenzyme A (AcAc-CoA, Cayman Chemicals)
were added to 40 mM MgCl₂ in PBS to give 400 μM final
concentration of each substrate. The substrate mixture was allowed
to preequilibrate at 37 °C, then mixed in a 1:1 ratio with the
reaction solutions, and imaged at 303 nm^{33 (link)} once a minute using a Synergy Neo HTS Microplate Reader (BioTek).
Relative AcAc-CoA consumption for each condition was calculated by
the normalized absolute difference between the 303 nm absorbance in
the ACAT1 containing reactions and the corresponding ACAT1-less control
reactions.
For MS/MS monitoring of Ac-CoA production, aliquots
of the reactions were taken at the indicated time points and mixed
with MeOH in a 4:1 MeOH/sample ratio to quench the enzymatic reaction.
Internal deuterated standards, 1 μL of 10 mM d3-serine, were
added to the extraction solution for sample normalization, the samples
were centrifuged for 15 min at 16,000g, and the supernatant
was kept for immediate LC–MS/MS analysis.

Malonic acid, 2-¹³C-Malonic acid, 4-coumaric acid, 2-methylthiazole-5-carboxylic acid, resveratrol, p-coumaric acid, 7-phenyl heptanoic acid, 6-phenyl hexanoic acid, 5-(4-flurophenyl)valeric acid, 4-biphenylacetic acid, N-acetyl-L-cysteine, benzoic acid adenosine triphosphate (ATP), coenzyme A trilithium salt (CoA), magnesium chloride, dimethyl sulfoxide (DMSO) hexahydrate, phospho(enol) pyruvate potassium salt, PEG 4000, NaCl, potassium and sodium phosphate, IPTG, kanamycin, penicillin, streptomycin, were purchased from Sigma-Aldrich. 2-Chloro-1,3-thiazole-5-carboxylic acid and 4-methylthiazole-5-carboxylic acid were obtained from Matrix Scientific.