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

Authors: Wang, X  Stanley, WC  Brunengraber, H  Kasumov, T  Kasumov, Takhar 
Citation: Wang X, etal., Anal Biochem. 2007 Apr 15;363(2):169-74. Epub 2007 Jan 13.
Pubmed: (View Article at PubMed) PMID:17316539
DOI: Full-text: DOI:10.1016/j.ab.2007.01.010

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-(13)C(3)]malonyl-CoA to form [U-(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 extracts. Newly formed [U-(13)C(2)]acetyl-CoA and internal standard of [(2)H(3),1-(13)C]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 202muM and V(max) of 3.3mumol min(-1) (g liver)(-1) were calculated. The liver MCD activities (mumol 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.


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RGD Object Information
RGD ID: 1600790
Created: 2007-03-27
Species: All species
Last Modified: 2007-03-27
Status: ACTIVE


RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.