Go to navigation (press enter key)Menu
Chemistry
Completed Project

Optimization of the in vitro Assay Conditions of the Arabidopsis thaliana Acyltransferase At1g78690

Domenic Pedulla, Vassar College ’16 and Professor Teresa A. Garrett
Headgroup-acylated glycerophospholipids (GPLs) are a class of lipids in the membrane of Escherichia coli with potentially important but not well- understood roles in membrane function. The in vivo overexpression of the Arabidopsis thaliana lyso-GPL acyltransferase At1g78690 leads to the accumulation of acyl phosphatidylglycerol, a headgroup-acylated GPL. However, previous work has shown that in vitro, this enzyme catalyzes the acylation of a variety of specific lyso-GPLs, lyso-phosphatidylglycerol, lyso-phosphatidylethanolamine (lyso-PE), lyso-phosphatidylinosital, and lyso-phosphatidylcholine, to their diacylated forms. In order to more fully understand the metabolic pathways linking the expression of a lyso-GPL acyltransferase to the formation of headgroup acylated GPLs, we must first optimize the in vitro reaction conditions for At1g78690. Using existing optimization data as guidelines, we sought to determine the optimal pH, detergent, and EGTA levels in the reaction mixture. Membranes isolated from E. coli overexpressing At1g78690 were reacted with a lyso PE acyl acceptor and a 14C-labeled palmitoyl Coenzyme A acyl donor under various reaction conditions. The extent of the acyl transfer reaction was monitored by resolving 14C-labeled products using thin layer chromatography. The percent of PE formed was used to determine the specific activity of the At1g78690 enzyme under each condition tested. The results of these assays suggest that the optimal pH of this reaction is between 7.0 and 7.4 and that Sodium cholate and Sodium deoxycholate detergents enhance the rate of reaction to the greatest extent. Combining this data with previous optimization data, we will generate a fully optimized in vitro enzyme assay for At1g78690 that will be used to perform an in-depth characterization of this enzyme’s substrate specificity.