Journal of Oil Palm Research Vol. Special Issue  2008 July p.  30-36
DOI:

Non-radioactive assay for acetyl-CoA carboxylase activity

Author(s): WILLIS, Laura Willis* ; WAN SARIDAH Wan Omar** ; SAMBANTHAMURTHI, Ravigadevi** ; SINSKEY, Anthony J*

Acetyl-CoA carboxylase is a key enzyme in oil biosynthesis and is critical for the oil deposition pathway. This biotinylated enzyme catalyzes the first committed step in fatty acid biosynthesis, the ligation of a carbon to acetyl-CoA to form malonyl-CoA. The acetyl-CoA carboxylase holoenzyme has four distinct protein domains: biotin carboxylase, biotin carboxylase carrier protein, and the alpha and beta transcarboxylase domains. Biotin carboxylase catalyzes the addition of carbon dioxide to biotin carboxylase carrier protein, while the alpha and beta subunits of carboxyltransferase ligate the activated CO2 to acetyl-CoA. The canonical assays for monitoring the activity of acetyl-CoA carboxylase rely on incorporation of radiolabelled acetyl-CoA. In this work, we describe the development of a discontinuous, non-radioactive spectrophotometric assay for acetyl-CoA carboxylase activity. Permeabilized Corynebacterium glutamicum cells were added to an assay mixture containing acetyl-CoA, bicarbonate, magnesium and ATP, and aliquots were removed at set time points and stopped by the addition of trifluoroacetic acid. The level of acetyl-CoA remaining in each aliquot was determined via a citrate synthase assay, in which the formation of the yellow compound dithiobisnitrobenzoic acidthiophenolate was followed spectrophotometrically at 412 nm.

Keywords: , , , ,

Author Information
* Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
E-mail: asinskey@mit.edu

** Malaysian Palm Oil Board, P. O. Box 10620, 50720 Kuala Lumpur, Malaysia.

Journal of Oil Palm Research Special Issue on Malaysia-MIT Biotechnology Partnership Programme: Volume 2 - Oil Palm Metabolic Engineering


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