Measurement of lysophospholipid acyltransferase activities using substrate competition

  • Posted on: 5 November 2014
  • By: fcoldren
TitleMeasurement of lysophospholipid acyltransferase activities using substrate competition
Publication TypeJournal Article
Year of Publication2014
AuthorsMartin SA, Gijón MA, Voelker DR, Murphy RC
JournalJ Lipid Res
Date Published2014 Apr

Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoA-dependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. We describe here an alternative biochemical assay that provides a mixture of substrates presented to the microsomal extracts. Microsomal preparations from RAW 264.7 cells were used to compare traditional LPAT assays with data obtained using a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using LC-MS/MS. Both types of assays provided similar results, but the dual choice assay provided information about multiple fatty acyl chain incorporation into various phospholipid classes in a single reaction. Engineered suppression of LPCAT3 activity in RAW 264.7 cells was easily detected by the dual choice method. These findings demonstrate that this assay is both specific and sensitive and that it provides much richer biochemical detail than traditional assays.

Alternate JournalJ. Lipid Res.
PubMed ID24563510
PubMed Central IDPMC3966712
Grant ListF31 NS080486 / NS / NINDS NIH HHS / United States
HL117798 / HL / NHLBI NIH HHS / United States
NS080486 / NS / NINDS NIH HHS / United States