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A Novel Assay of DGAT Activity Based on High Temperature GC/MS of Triacylglycerol
Author(s) -
Greer Michael S.,
Zhou Ting,
Weselake Randall J.
Publication year - 2014
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-014-3921-8
Subject(s) - microsome , acyl coa , chemistry , biochemistry , substrate (aquarium) , enzyme , chromatography , diacylglycerol kinase , acyltransferases , specific activity , enzyme assay , glycerol , mass spectrometry , transferase , biosynthesis , biology , ecology , protein kinase c
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl‐CoA‐dependent biosynthesis of triacylglycerol (TAG), a high‐energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high‐temperature gas chromatography (GC)/mass spectrometry (MS)‐based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type‐1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS‐based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1‐ 14 C] acyl‐CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.