Assay of the activity of malonyl–coenzyme A decarboxylase by gas chromatography–mass spectrometry

We developed a gas chromatography–mass spectrometry (GC–MS) assay to measure the activity of malonyl–coenzyme A (CoA) decarboxylase (MCD) in crude tissue homogenates. Liver extracts are incubated with [U-13C3]malonyl–CoA to form [U-13C2]acetyl–CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-13C2]acetyl–CoA by acetyl–CoA hydrolase present in the extracts. Newly formed [U-13C2]acetyl–CoA and internal standard of [2H3,1-13C]acetyl–CoA are analyzed as thiophenol derivatives by GC–MS. This assay was applied to a study of the kinetics of MCD in rat liver. Using the Lineweaver–Burke plot of MCD kinetics, Km of 202 μM and Vmax of 3.3 μmol min−1 (g liver)−1 were calculated. The liver MCD activities (μmol min−1 g−1 ± SD) in three groups of rats with different nutritional statuses—fed, 1-day fasted, and 2-day fasted—were 1.80 ± 0.41, 2.59 ± 0.37 (P < 0.05), and 3.07 ± 0.70 (P < 0.05), respectively. We report a practical, nonradioactive, sensitive assay of MCD in crude tissue extract.