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-(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 202microM and V(max) of 3.3micromol min(-1) (g liver)(-1) were calculated. The liver MCD activities (micromol 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.
Wang
Xiao
X
Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA.
Stanley
William C
WC
Darrow
Cory J
CJ
Brunengraber
Henri
H
Kasumov
Takhar
T
D000818:Animals; D002262:Carboxy-Lyases / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D014020:Tissue Extracts
D014020:Tissue Extracts; D008316:Malonyl Coenzyme A; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase