A Fluorescence‐Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity. Issue 9 (2nd September 2019)

• Record Type: Journal Article
• Authors: Falarz, Lucas; Xu, Yang; Singer, Stacy D.; Chen, Guanqun
• Is Part Of: Lipids. Volume 54:Issue 9(2019)
• Page Start: 571
• Page End: 579
• Publication Date: 2019-09-02
• Subjects: Fluorescent diacylglycerol -- in vitro assay -- Nitrobenzoxadiazole -- PDAT -- Triacylglycerol biosynthesis; Lipids -- Periodicals; Lipids -- Periodicals; Lipiden; Lipides -- Périodiques; 547.77
• DOI: 10.1002/lipd.12190
• Languages: English
• ISSNs: 0024-4201

Abstract

Abstract: 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.