Formation of vesicles by the action of acyl-CoA:1-acyllsophosphatidylcholine acyltransferase from rat liver microsomes: optimal solubilization conditions and analysis of lipid composition and enzyme activity.

Abstract

The enzyme acyl coenzyme A:1-acyllysophosphatidylcholine acyltransferase (acyl-CoA:lysoPC acyltransferase) can be isolated in newly formed phosphatidylcholine (PC) vesicles by solubilization of rat liver microsomes with the two substrates lysoPC and acyl-CoA. In this study, we sought to optimized the conditions for the formation of PC vesicles and analyzed the lipid composition and enzyme activity of the newly formed vesicles. Analysis of PC vesicles formed by incubation of the microsomal preparation with 1-(C16:0)lysoPC and C18:1CoA, C18:2CoA, or C20:4CoA showed that the optimal protein:lysoPC ratio was 1:5 (by weight) and the optimal lysoPC:acyl-CoA ratio was 1:1 (molar amounts). PC formation increased with incubation time; after 20 h of incubation at 37 degrees C, approximately 75% of the lysoPC was converted to PC in the incubation mixture. The phospholipid molecular species composition of the vesicles reflected almost exclusively the substrates used; the vesicles contained approximately 33% of the total acyl-CoA:lysoPC acyltransferase activity from the microsomes and demonstrated a single protein band with a molecular mass of 21 kDa by gel electrophoresis. The procedure selected for the enzyme specific for lysoPC acylation, as enzyme activity toward lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), and lysophosphatidylinositol (lysoPI), was very low. In addition, the utilization of different acyl-CoA substrates for acylation of lysoPC was different from that in microsomes. These results show that an enzyme specific for the formation of PC from lysoPC can be isolated in PC vesicles with a designed phospholipid molecular species composition and that the lipid environment plays an important role in the regulation of the enzyme's affinity for its substrates.