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A Fluorescence‐Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity
Author(s) -
Falarz Lucas,
Xu Yang,
Singer Stacy D.,
Chen Guanqun
Publication year - 2019
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1002/lipd.12190
Subject(s) - biochemistry , acyltransferase , diacylglycerol kinase , phospholipid , arabidopsis thaliana , enzyme , recombinant dna , biosynthesis , chemistry , biology , acyltransferases , substrate (aquarium) , mutant , gene , ecology , protein kinase c , membrane
Phospholipid:diacylglycerol acyltransferase (PDAT) catalyzes the acyl‐CoA‐independent triacylglycerol (TAG) biosynthesis in plants and oleaginous microorganisms and thus is a key target in lipid research. The conventional in vitro PDAT activity assay involves the use of radiolabeled substrates, which, however, are expensive and demand strict regulation. In this study, a reliable fluorescence‐based method using nitrobenzoxadiazole‐labeled diacylglycerol (NBD‐DAG) as an alternative substrate was established and subsequently used to characterize the enzyme activity and kinetics of a recombinant Arabidopsis thaliana PDAT1 (AtPDAT1). We also demonstrate that the highly toxic benzene used in typical PDAT assays can be substituted with diethyl ether without affecting the formation rate of NBD‐TAG. Overall, this method works well with a broad range of PDAT protein content and shows linear correlation with the conventional method with radiolabeled substrates, and thus may be applicable to PDAT from various plant and microorganism species.