A Fluorescence‐Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity

• Author(s) / Creator(s)

  • Falarz, Lucas
  • Xu, Yang
  • Singer, Stacy D.
  • Chen, Guanqun

• 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.

• Date created

  2019-09-02

• Subjects / Keywords

  • in vitro assay
  • PDAT
  • fluorescent diacylglycerol
  • nitrobenzoxadiazole
  • triacylglycerol biosynthesis

• Type of Item

  Article (Draft / Submitted)

• DOI

  https://doi.org/10.7939/r3-8k2j-tf81

• License

  This is the peer reviewed version of the following article: Falarz, L., Xu, Y., Singer, S. D., & Chen, G. (2019). A Fluorescence-Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity. Lipids, 54(9), 571–579. , which has been published in final form at https://doi.org/10.1002/lipd.12190. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.