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A NOVEL ASSAY OF ACYL‐COA:DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY UTILIZING FLUORESCENT SUBSTRATE 1
Author(s) -
Sanderson Matthew C.,
Venable Mark E.
Publication year - 2012
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.1529-8817.2012.01137.x
Subject(s) - diacylglycerol kinase , biology , acyl coa , acyltransferases , biochemistry , microsome , substrate (aquarium) , enzyme , substrate specificity , enzyme assay , acyltransferase , endogeny , biosynthesis , ecology , protein kinase c
Common methods for assaying acyl‐CoA:diacylglycerol acyltransferase (DGAT) enzymatic activity rely upon radiolabeled substrates or product assay. We developed a novel assay that directly quantifies endogenous DGAT activity through the use of a fluorescently labeled substrate. We performed this assay with microsomal protein, 2‐(6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)hexanoyl‐1‐hexadecanoyl‐ sn ‐glycero‐3‐diacylglycerol (NBD‐DAG), and oleoyl‐CoA substrates. DGAT activity was analyzed in three species of algae as well as rat liver. The protocol proved to be sensitive and reliable. This assay may be used to facilitate research in the areas of biodiesel, oilseed crops, and triacylglycerol‐related human pathologies.