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Assay of the activity of malonyl‐CoA decarboxylase (MCD) by gas chromatography‐mass spectrometry (GC‐MS)
Author(s) -
Wang Xiao,
Brunengraber Henri,
Kasumov Takhar
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a139-b
Subject(s) - chemistry , gas chromatography–mass spectrometry , hydrolysis , chromatography , mass spectrometry , ammonia , acetyl coa , biochemistry , enzyme
We developed a GC‐MS assay for the activity of MCD in tissue homogenates. Extracts of rat liver (0.25 mg tissue equivalent/assay) are incubated at 37°C with 0.4 mM [U‐ 13 C 3 ]malonyl‐CoA to form [1,2‐ 13 C 2 ]acetyl‐CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2‐ 13 C 2 ]acetyl‐CoA by acetyl‐CoA hydrolase present in the tissue extracts. After stopping the reaction with methanol, an internal standard of [ 2 H 3 , 1‐ 13 C]acetyl‐CoA is added before reacting the two forms of acetyl‐CoA with thiophenol. The product acetylthiophenol is analyzed by ammonia positive chemical ionization GC‐MS, monitoring 170 (M + NH 4 +) to 174 (M + NH 4 + + 4). The assay was applied to a study of the kinetics of MCD in rat liver, yielding K m and V max of 171 μM and 2.5 μmol·min 1 ·(g liver) 1 , respectively. Also, 10 μM CBM‐301940, a potent MCD inhibitor (Chugai Pharma), decreased MCD activity 7‐fold. Lastly, liver MCD activities (μmol·min 1 ·g 1 ± S.D.) in three groups of rats that were fed, one‐day fasted or two‐day fasted were 1.66 ± 0.53, 2.35 ± 0.38 (p < 0.05), and 2.72 ± 0.62 (p < 0.05) respectively. Supported by NIH Roadmap grant R33DK070291.