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.
Highlights
Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoAdependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells
Since 2005, several groups have performed extensive analysis of the function of these enzymes and discovered that LPATs belong to two different protein families, represented by the lysophosphatidic acid acyltransferases (LPAATs) and the membrane bound O-acyltransferases (MBOATs) [5,6,7,8,9,10,11,12,13,14,15,16]
We describe a dual substrate choice assay using microsomal extracts containing LPATs expressed in mammalian cells to test the activity of these proteins in a single experimental model and compare the results with the more traditional means of determining substrate specificity and enzymatic rate
Summary
Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoAdependent 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. 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 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.—Martin, S. These families both contain membrane-bound proteins that have not been purified from tissues [17], but their encoding cDNAs have been cloned and expressed in a variety of host cells including yeast [10, 13, 18]
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