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

Abstract

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- 13C 3]malonyl–CoA to form [U- 13C 2]acetyl–CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2- 13C 2]acetyl–CoA by acetyl–CoA hydrolase present in the extracts. Newly formed [U- 13C 2]acetyl–CoA and internal standard of [ 2H 3,1- 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, K m of 202 μM and V max 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.