Measurement of lysophospholipid acyltransferase activities using substrate competition
Title | Measurement of lysophospholipid acyltransferase activities using substrate competition |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Martin SA, Gijón MA, Voelker DR, Murphy RC |
Journal | J Lipid Res |
Volume | 55 |
Issue | 4 |
Pagination | 782-91 |
Date Published | 2014 Apr |
ISSN | 0022-2275 |
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. |
DOI | 10.1194/jlr.D044636 |
Alternate Journal | J. Lipid Res. |
PubMed ID | 24563510 |
PubMed Central ID | PMC3966712 |
Grant List | F31 NS080486 / NS / NINDS NIH HHS / United States HL117798 / HL / NHLBI NIH HHS / United States NS080486 / NS / NINDS NIH HHS / United States |