Dataset Information

Measurement of lysophospholipid acyltransferase activities using substrate competition.

ABSTRACT: 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.

SUBMITTER: Martin SA

PROVIDER: S-EPMC3966712 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Measurement of lysophospholipid acyltransferase activities using substrate competition.

Martin Sarah A SA Gijón Miguel A MA Voelker Dennis R DR Murphy Robert C RC

Journal of lipid research 20140221 4

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 mixtur ...[more]

PMID: 24563510

Similar Datasets

Project description:N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38:6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38:6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44:12)] and DHA-DHA-PE [PE(44:12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.

Dataset Information

Measurement of lysophospholipid acyltransferase activities using substrate competition.

Publications

Measurement of lysophospholipid acyltransferase activities using substrate competition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets